Spinner preparation machine and cavity resonator

ABSTRACT

The present invention relates to the treatment of disorders using heme oxygenase-1 and heme degradation products.

CLAIM OF PRIORITY

This application claims priority under 35 USC §119(e) to U.S. PatentApplication Ser. No. 60/372,762, filed on Apr. 15, 2002, the entirecontents of which are hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No. HL58688awarded by the National Institutes of Health. The Government has certainrights in the invention.

TECHNICAL FIELD

The present invention relates to the treatment of disorders using hemeoxygenase-1 and heme degradation products.

BACKGROUND

Heme oxygenase-1 (HO-1) catalyzes the first step in the degradation ofheme. HO-1 cleaves the α-meso carbon bridge of b-type heme molecules byoxidation to yield equimolar quantities of biliverdin IXa, carbonmonoxide (CO), and free iron. Subsequently, biliverdin is converted tobilirubin via biliverdin reductase, and the release of Fe²⁺ from hemeinduces the expression of the Fe²⁺ sequestering protein ferritin, whichacts as an anti-oxidant by limiting the ability of Fe²⁺ to participatein the generation of free radicals through the Fenton reaction.

SUMMARY

The present invention is based, in part, on the discovery that theadministration of degradation products of heme and/or HO-1 can attenuateinflammation and suppress the damage associated with ischemia.

Accordingly, the present invention features a method of reducinginflammation in a patient that includes identifying a patient sufferingfrom or at risk for inflammation, and administering to the patient atreatment including inducing ferritin in the patient; expressingferritin in the patient; and/or administering a pharmaceuticalcomposition comprising HO-1, bilirubin, biliverdin, ferritin, iron,desferoxamine (DFO), salicylaldehyde isonicotinoyl hydrazone (SIH), irondextran, and/or apoferritin to the patient, in an amount sufficient toreduce inflammation.

In one embodiment, the treatment is administering a pharmaceuticalcomposition that includes biliverdin. The pharmaceutical composition canbe administered to the patient at a dosage of, for example, about 1 to1000 micromoles/kg/day, e.g., 10 to 500 μmols/kg/day, 20 to 200μmols/kg/day, or 25 to 100 μmols/kg/day.

Alternatively or in addition, the treatment can include administering apharmaceutical composition that includes bilirubin to the patient. Thepharmaceutical composition can be administered to a patient to generateserum levels of bilirubin in a range of from about 1 to about 300μmols/L, e.g., about 10 to about 200 μmols/L, or about 50 to about 100μmols/L. Individual doses of bilirubin can be administered, which canfall within the range of about 1 to 1000 mg/kg, e.g., 10 to 500 mg/kg,20 to 200 mg/kg, or 25 to 150 mg/kg.

Further, the treatment can include administering a pharmaceuticalcomposition that includes apoferritin to the patient. The pharmaceuticalcomposition can be administered to the patient at a dosage of, forexample, about 1 to 1000 mg/kg, e.g., 10 to 500 mg/kg, 20 to 200 mg/kg,or 25 to 150 mg/kg.

The treatment can also include administering a pharmaceuticalcomposition that includes DFO to the patient. The pharmaceuticalcomposition can be administered to the patient at a dosage of, forexample, about 0.1 to 1000 mg/kg, e.g., 0.5 to 800 mg/kg, 1 to 600mg/kg, 2 to 400 mg/kg, or 2.5 to 250 mg/kg.

Further, the treatment can include administering a pharmaceuticalcomposition that includes iron dextran to the patient. Thepharmaceutical composition can be administered to the patient at adosage of, for example, about 1 to 1000 mg/kg, e.g., 10 to 900 mg/kg,100 to 800 mg/kg, 300 to 700 mg/kg, or 400 to 600 mg/kg. Alternatively,free iron, e.g., in the form of iron supplements, can be delivered tothe patient.

The treatment can also include administering a pharmaceuticalcomposition that includes salicylaldehyde isonicotinoyl hydrazone (SIH)to the patient. The pharmaceutical composition can be administered tothe patient orally or parenterally at a dosage of, for example, about0.02 to 100 mmol/kg, e.g., about 0.02 to 10 mmol/kg, e.g., 0.02 to 50mmol/kg, or 0.2 to 20 mmol/kg.

The inflammation can be associated with a condition including, but notlimited to, asthma, adult respiratory distress syndrome, interstitialpulmonary fibrosis, pulmonary emboli, chronic obstructive pulmonarydisease, primary pulmonary hypertension, chronic pulmonary emphysema,congestive heart failure, peripheral vascular disease, stroke,atherosclerosis, ischemia-reperfusion injury, heart attacks,glomerulonephritis, conditions involving inflammation of the kidney,infection of the genitourinary tract, viral and toxic hepatitis,cirrhosis, ileus, necrotizing enterocolitis, specific and non-specificinflammatory bowel disease, rheumatoid arthritis, deficient woundhealing, Alzheimer's disease, Parkinson's disease, graft versus hostdisease, or hemorrhagic, septic, or anaphylactic shock.

In an embodiment of the present invention, the inflammation isinflammation of the heart, lung, liver, pancreas, joints, eye, bronchi,spleen, brain, skin, and/or kidney. The inflammation can also be aninflammatory condition localized in the gastrointestinal tract. Theinflammatory condition can be, for example, amoebic dysentery, bacillarydysentery, schistosomiasis, campylobacter enterocolitis, yersiniaenterocolitis, enterobius vermicularis, radiation enterocolitis,ischaemic colitis, eosinophilic gastroenteritis, ulcerative colitis,indeterminate colitis, and Crohn's disease.

The method can further include the step(s) of inducing and/or expressingHO-1 in the patient and/or administering a pharmaceutical compositioncomprising carbon monoxide to the patient.

In another aspect, the invention features a method of transplanting anorgan, which includes administering to a donor a treatment includinginducing HO-1 or ferritin in the donor; expressing ferritin in thedonor; and/or administering a pharmaceutical composition comprisingHO-1, bilirubin, biliverdin, ferritin, desferoxamine, salicylaldehydeisonicotinoyl hydrazone, iron dextran, and/or apoferritin to the donor;obtaining an organ from the donor; and transplanting the organ into arecipient, wherein the treatment administered is sufficient to enhancesurvival or function of the organ after transplantation into therecipient. In certain embodiments, the method further includes thestep(s) of inducing and/or expressing HO-1 in the donor and/oradministering a pharmaceutical composition comprising carbon monoxide tothe donor.

The invention also features a method of transplanting an organ, whichincludes providing an organ of a donor; administering to the organ exvivo a treatment including inducing HO-1 or ferritin in the organ,expressing HO-1 or ferritin in the organ, and/or administering apharmaceutical composition comprising HO-1, bilirubin, biliverdin,ferritin, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, irondextran, and/or apoferritin; and transplanting the organ into arecipient, wherein treatment administered to the organ is sufficient toenhance survival or function of the organ after transplantation. Incertain embodiments, the method further includes the step(s) of inducingand/or expressing HO-1 in the organ and/or administering apharmaceutical composition comprising carbon monoxide to the organ.

Further, the invention features a method of transplanting an organ,which includes providing an organ from a donor; transplanting the organinto a recipient; and before, during, or after the transplanting step,administering to the recipient a treatment including inducing HO-1 orferritin in the recipient, expressing HO-1 or ferritin in the recipient,and/or administering a pharmaceutical composition comprising HO-1,bilirubin, biliverdin, ferritin, desferoxamine, salicylaldehydeisonicotinoyl hydrazone, iron dextran, and/or apoferritin to therecipient, wherein the treatment administered to the recipient issufficient to enhance survival or function of the organ aftertransplantation of the organ to the recipient. In certain embodiments,the method further includes the step(s) of inducing and/or expressingHO-1 in the recipient and/or administering a pharmaceutical compositioncomprising carbon monoxide to the recipient.

In another aspect, the invention provides a method of performingangioplasty on a patient, which includes performing angioplasty on thepatient; and before, during, or after the performing step, administeringa treatment including inducing HO-1 or ferritin in the patient,expressing HO-1 or ferritin in the patient, and/or administering apharmaceutical composition comprising HO-1, bilirubin, biliverdin,ferritin, desferoxamine, salicylaldehyde isonicotinoyl hydrazone, irondextran, and/or apoferritin to the patient. In certain embodiments, themethod further includes the step(s) of inducing and/or expressing HO-1in the patient and/or administering a pharmaceutical compositioncomprising carbon monoxide to the patient.

The invention also features a method of performing vascular surgery on apatient, which includes performing vascular surgery on the patient; andbefore, during, or after performing the vascular surgery, administeringto the patient at least one treatment including inducing HO-1 orferritin in the patient, expressing ferritin in the patient, and/oradministering a pharmaceutical composition comprising HO-1, bilirubin,biliverdin, ferritin, desferoxamine, salicylaldehyde isonicotinoylhydrazone, iron dextran, and/or apoferritin. In certain embodiments, themethod includes the step(s) of inducing and/or expressing HO-1 in thepatient and/or administering a pharmaceutical composition comprisingcarbon monoxide to the patient.

In yet another aspect, the invention features a method of treating acellular proliferative and/or differentiative disorder in a patient,which includes identifying a patient suffering from or at risk for acellular proliferative and/or differentiative disorder; andadministering to the patient at least one treatment including inducingferritin in the patient, expressing ferritin in the patient, and/oradministering a pharmaceutical composition comprising HO-1, bilirubin,biliverdin, ferritin, iron, desferoxamine, salicylaldehyde isonicotinoylhydrazone, iron dextran, and/or apoferritin to the patient, in an amountsufficient to treat the cellular proliferative and/or differentiativedisorder. In certain embodiments, the method further includes thestep(s) of inducing and/or expressing HO-1 in the patient and/oradministering a pharmaceutical composition comprising carbon monoxide tothe patient.

In still another aspect, the invention features a method of reducing theeffects of ischemia in a patient, which includes identifying a patientsuffering from or at risk for ischemia; and

administering to the patient at least one treatment including inducingferritin in the patient, expressing ferritin in the patient, and/oradministering a pharmaceutical composition comprising HO-1, bilirubin,biliverdin, ferritin, iron, desferoxamine, salicylaldehyde isonicotinoylhydrazone, iron dextran, and/or apoferritin to the patient, in an amountsufficient to reduce the effects of ischemia. In certain embodiments,the method further includes the step(s) of inducing and/or expressingHO-1 in the patient and/or administering a pharmaceutical compositioncomprising carbon monoxide to the patient.

The term “pharmaceutical composition” is used throughout thespecification to describe a gaseous, liquid, or solid compositioncontaining an active ingredient, e.g., HO-1 or ferritin (or an inducerof HO-1 or ferritin), bilirubin, and/or biliverdin, that can beadministered to a patient and/or an organ. The invention contemplatesuse of any two, three, four, or five of these in combination or insequence. The skilled practitioner will recognize which form of thepharmaceutical composition, e.g., gaseous, liquid, and/or solid, ispreferred for a given application. Further, the skilled practitionerwill recognize which active ingredient(s) should be included in thepharmaceutical composition for a given application.

The term “patient” is used throughout the specification to describe ananimal, human or non-human, to whom treatment according to the methodsof the present invention is provided. Veterinary applications areclearly anticipated by the present invention. The term includes but isnot limited to mammals, e.g., humans, other primates, pigs, rodents suchas mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats,dogs, sheep and goats.

The terms “effective amount” and “effective to treat,” as used herein,refer to the administration of a pharmaceutical compositions(s)described herein in an amount or concentration and for period of timeincluding acute or chronic administration and periodic or continuousadministration that is effective within the context of itsadministration for causing an intended effect or physiological outcome.The terms “treat” or “treatment,” are used herein to describe delayingthe onset of, inhibiting, or alleviating the effects of a disease orcondition, e.g., a disease or condition described herein.

Also within the invention is the use of HO-1 and/or any of thedegradation products of heme, e.g., bilirubin, biliverdin, ferritin,iron, desferoxamine (DFO), salicylaldehyde isonicotinoyl hydrazone(SIH), iron dextran, and/or apoferritin, in the manufacture of amedicament for the treatment or prevention of inflammation or the damageassociated with ischemia, e.g., transplantation-related ischemia. Themedicament can be used in a method for treating or preventinginflammation in a patient suffering from or at risk for inflammation.The medicament can also be used in a method of organ transplantation,e.g., to reduce inflammation and ischemia-reperfusion injury. Themedicament can also be used in a method of performing vascular surgeryor angioplasty on a patient. The medicament can also be used in a methodof treating a cellular proliferative and/or differentiative disorder ina patient. The medicament can also be used in a method of treating orpreventing the effects of ischemia in a patient. The medicament can bein any form described herein, and can be administered alone or incombination with, e.g., CO.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photograph of a gel showing the results ofsemi-quantitative PCR analysis of HO-1 and β-actin mRNA levels afterinduction of DSS-colitis in control animals.

FIG. 1B is a bar graph illustrating the changing ratio of HO-1: β-actinmRNA levels after induction of DSS-colitis in control animals. X-axis:days.

FIG. 1C is a photograph of a Western blot showing an increase in HO-1protein levels after induction of DSS-colitis in control animals.α-tubulin was used as a internal reference.

FIG. 2A is a photograph of a gel showing the results ofsemi-quantitative PCR analysis of HO-1 and β-actin mRNA levels afterinduction of DSS-colitis in animals treated with cobalt protoporphyrin(CoPP).

FIG. 2B is a bar graph illustrating the changing ratio of HO-1: β-actinmRNA levels after induction of DSS-colitis in animals treated with CoPP.

FIG. 2C is a photograph of a Western blot showing an increase in HO-1protein levels after induction of DSS-colitis in animals treated withCoPP. α-tubulin was used as a loading control.

FIG. 3A is a bar graph that illustrates the effect of cobaltprotoporphyrin treatment on weight loss associated with DSS-colitis, asobserved on day 7 of the experiment. NT=no treatment; CoPP=cobaltprotoporphyrin; ZNPP=zinc protoporphyrin.

FIG. 3B is a bar graph that illustrates the effect of cobaltprotoporphyrin treatment on the DSS-colitis disease activity index(DAI), as observed on day 7 of the experiment.

FIG. 3C is a line graph that illustrates the effect of cobaltprotoporphyrin treatment on the DSS-colitis DAI over a period of 7 days.

FIG. 3D is a line graph that illustrates the effect of cobalt treatmenton intestinal bleeding associated with DSS-colitis over a period of 7days.

FIG. 3E is a line graph that illustrates the effect of cobaltprotoporphyrin induction of HO-1 on stool abnormalities associated withDSS-colitis over a period of 7 days.

FIG. 4A is a set of four photomicrographs of cryptal structures at 10×(top row) and 40× (bottom row) magnification in untreated controlanimals (NT, left column) and CoPP-treated animals (COPP, right column).

FIG. 4B is a bar graph illustrating the relatively decreased damage tomucosal glands in animals treated with CoPP as compared to untreatedcontrols, as measured by crypt scores.

FIG. 5A is a bar graph that illustrates the effect of treatment withbiliverdin, DFO or carbon monoxide on weight loss associated withDSS-colitis, as observed on day 7 of the experiment. NT=no treatment;CO=carbon monoxide; DFO=desferoxamine.

FIG. 5B is a bar graph that illustrates the effect of treatment withbiliverdin, DFO or carbon monoxide on the DSS-colitis disease activityindex (DAI), as observed on day 7 of the experiment.

FIG. 5C is a line graph that illustrates the effect of treatment withbiliverdin on the DSS-colitis DAI over a period of 7 days.

FIG. 5D is a line graph that illustrates the effect of treatment withbiliverdin on intestinal bleeding associated with DSS-colitis over aperiod of 7 days.

FIG. 5E is a line graph that illustrates the effect of treatment withbiliverdin on stool abnormalities associated with DSS-colitis over aperiod of 7 days.

FIG. 6A is a set of four photomicrographs of cryptal structures at 10×(top row) and 40× (bottom row) magnification in untreated controlanimals (NT, left column) and biliverdin-treated animals (right column).

FIG. 6B is a bar graph illustrating the relatively decreased damage tomucosal glands in animals treated with biliverdin as compared tountreated controls, as measured by crypt scores.

FIG. 7A is a set of photographs showing the results of Western blotanalysis of HO-1 (top row) and α-actin (bottom row) protein expressionin spleen (left column) and heart (right column) at 0, 1, 2, 4, and 7days after CoPPIX administration.

FIG. 7B is a bar graph illustrating the increase in HO-1 expressionlevels at 0, 1, 2, 4, and 7 days after CoPPIX administration.

FIG. 7C is a bar graph illustrating the effect of treatment with CoPPIXor ZnPPIX on bilirubin levels.

FIG. 7D is a line graph illustrating the percent survival of cardiacallografts in animals treated with CoPPIX or ZNPPIX.

FIG. 8A is a line graph illustrating the % survival of cardiacallografts in animals treated with biliverdin on three different dosageschedules.

FIG. 8B is a line graph illustrating the increase in serum bilirubinlevels with time after administration of biliverdin.

FIG. 8C is a line graph illustrating percent survival in mice challengedby second set transplantation using cardiac allografts from donor(DBA/2J) or third party (FVB) mouse strain.

FIG. 9 is a bar graph illustrating the effect on proliferation ofsplenocytes in aniinals treated with CoPPIX, ZNPPIX, orbiliverdin(1×/day or 3×/day).

FIG. 10A is a bar graph illustrating the effect of different doses ofbiliverdin on proliferation of splenocytes stimulated with ConA.

FIG. 10B is a bar graph illustrating the effect of different doses ofbiliverdin on proliferation of splenocytes stimulated with anti-CD3 mAb.

FIG. 10C is abar graph illustrating the effect of different doses ofbiliverdin on proliferation of splenocytes stimulated with irradiatedDBA/2 splenocytes.

FIG. 10D is a bar graph illustrating the effect of different doses ofbiliverdin on proliferation of splenocytes co-stimulated with anti-CD3mAb plus anti-CD28.

FIG. 11A is a bar graph illustrating the suppressive effect of differentdoses of biliverdin on IL-2 secretion by splenocytes.

FIG. 111B is a bar graph illustrating the effect of adding exogenousIL-2 on the suppressive effect of different doses of biliverdin on IL-2secretion by splenocytes.

FIG. 11C is a panel of three graphs showing the results of FACS analysisof CD25 expression in splenocytes stimulated with anti-CD3 mAb oranti-CD3 mAb plus biliverdin.

FIG. 11D is a pair of photographs illustrating the effect of biliverdinon nuclear translocation of NF-κB or NFAT, as measured by DNA-binding.

FIG. 12A is a line graph illustrating percent graft survival in animalstreated with CoPP or ZnPP alone (donor treated on day-2 (D-2) and day-1(D-1), recipient treated from day-1 (D-1) to day +13 (D13)), DSI alone(recipient treated at day 0 (D0)), or CoPP or ZnPP (donor treated on D-2and D-1, recipient treated from D-1 to D13) plus DSI (recipient treatedat D0).

FIG. 12B is a line graph illustrating percent graft survival in animalstreated with CoPP or ZnPP alone (donor treated on day-2 (D-2) and day-1(D-1), recipient treated from day-8 (D-8) to day +6 (D6)), DST alone(recipient treated at D-7), or CoPP or ZnPP (donor treated for D-2 andD-1, recipient treated from D-8 to D6) plus DSI (recipient treated atday-7 (D-7)).

FIG. 13A is a pair of bar graphs illustrating percent cell survival inBAEC (left panel) or murine 2F-2B EC (ATCC; right panel) cotransfectedwith β-galactosidase plus control (pcDNA3 or pcDNA3/HO-1) orpcDNA3/H-Ferritin. Black bars indicate EC treated with INF-α and Act.D.Gray bars represent EC treated with Act.D. One representative experimentout of six is shown. All results shown are the mean±SD from duplicatewells.

FIG. 13B is a pair of bar graphs illustrating percent cell survival inmurine 2F-2B cells cotransfected with β-galactosidase plus control(pcDNA3) or pcDNA3/H-ferritin. Gray bars represent untreated EC andblack bars represent EC treated with etoposide (200 μM, 8 h; left panel)or subjected to serum deprivation (0.1% FCS, 24 hours; right panel). Onerepresentative experiment out of three is shown. All results shown arethe mean±SD from duplicate wells.

FIG. 13C is a bar graph illustrating percent cell survival in murine2F-2B cells cotransfected with β-galactosidase plus increasing amountsof pcDNA/H-ferritin as indicated. Cells transfected with pcDNA3 orpcDNA3/HO-1 were used as controls. Black bars indicate EC treated withTNF-α and Act.D. Gray bars represent EC treated with Act.D. Onerepresentative experiment out of three is shown. All results shown arethe mean±SD from duplicate wells.

FIG. 14A is a bar graph illustrating bile production in livers harvestedfrom SD rats, exposed to ischemia (24 hours, 4° C., UW solution) andperfused ex-vivo with syngeneic blood. Bile production is shown asmean±standard deviation from n=4. Notice that bile production at 120minutes was significantly higher in livers transduced with H-ferritinversus non-transduced (p<0.001) or β-galactosidase transduced livers(p<0.02).

FIG. 14B is a bar graph illustrating recipient survival in a KaplanMayer format. Livers transduced with control β-galactosidase orH-ferritin adenoviruses were transplanted into syngeneic recipients.Eight to ten animals were analyzed per group. Prolonged survival inrecipients receiving H-ferritin recombinant adenovirus transduced liverswas significantly enhanced as compared to recipients transplanted withnon-transduced or β-galactosidase recombinant adenovirus transducedlivers (p<0.001).

FIG. 14C is a set of photomicrographs of apoptosis in non-transducedlivers or livers transduced with control α-galactosidase or H-ferritinadenoviruses transplanted into syngeneic recipients. Apoptosis wasdetected 24 hours after transplantation as described herein. Apoptoticcells are signaled with black arrows. Magnifications are 100× (a, c, e)or 400× (b, d, f).

FIG. 15 is a schematic illustration of a model for the cytoprotectiveaction of ferritin. Top panel: control; bottom panel: in cellstransduced with recombinant H-ferritin adenovirus. SOD: superoxidedismutase.

FIG. 16 is a line graph illustrating the effect on islet transplantsurvival of treatment of the donor, recipient, or both with CoPP.

FIG. 17 is a line graph illustrating the effect on islet transplantsurvival of treatment of the donor and recipient with bilirubin orbiliverdin.

FIG. 18 is a pair of photomicrographs illustrating the effect ofpre-treatment with biliverdin on neointimal formation in rat carotidarteries after balloon injury.

FIG. 19 is a bar graph illustrating the effect of biliverdinpre-treatment on LPS-induced TNF-α production in rats. Ctl=control FIG.20 is a bar graph illustrating the effect of biliverdin pre-treatment onneutrophil accumulation in the lungs of rats treated with LPS. Inuntreated control rats, 200 of 200 cells are macrophages, no neutrophilsare present.

FIG. 21 is a bar graph illustrating the effect of biliverdinpre-treatment on protein accumulation in the lungs of rats treated withLPS. Normal baseline levels are around 30.

FIG. 22 is a bar graph illustrating the effect of biliverdinpre-treatment on IL-10 levels in rats treated with LPS.

FIGS. 23A-23D are bar graphs illustrating the effect of treatment withbiliverdin on levels of IL-6, IL-10, TNFα and IL-1β, respectively, in asmall intestine transplantation model. Real time RT-PCR analysisrevealed a significant increase in mRNA expression of IL-6, IL-10, TNF-αand IL-1b in graft muscularis 4 hours after transplantation compared tounoperated animals. In the recipients treated with CO, the meanexpressions for IL-6 and IL-1b mRNA, but not IL-10 or TNF-α, weresignificantly reduced. NM=Normal; BV=biliverdin; SITx-small intestinetransplant FIGS. 24A-24D are bar graphs illustrating the effect oftreatment with biliverdin on levels of COX-2; iNOS; ICAM-1, and MnSOD,respectively, in a small intestine transplantation model. INOS and COX-2gene expression was significantly upregulated in the muscularis of thetransplanted intestine. BV treatment significantly reduced theexpression of iNOS, COX-2, ICAM-1 and MnSOD mRNA in the graft muscularisextracts.

FIG. 25 is a line graph illustrating the effect of treatment withincreasing doses of bethanechol on contractility response intransplanted small intestine. Jejunal circular muscle strips fromcontrol animals (filled triangles) and unoperated animals receiving BVtreatment (filled squares) showed a dose-dependent increase incontractile area in response to bethanechol. This activity wassignificantly diminished in graft muscle taken 24 hours followingtransplantation (open triangles). Significant improvement was measuredin transplanted animals treated with BV (open squares) (N=5 each).

FIGS. 26A-26B are bar graphs illustrating the effect of treatment withbiliverdin on permeability and blood flow, respectively, in a smallintestine transplant model.

FIG. 27 is a line graph illustrating the effect of treatment withbiliverdin on antioxidant capacity in a small intestine transplantmodel.

DETAILED DESCRIPTION

The present invention includes providing, e.g., administering, inducingand/or expressing any or all of the products of heme degradation in apatient to treat various diseases or conditions, e.g., inflammation,and/or to improve the outcome of various surgical procedures, e.g.,transplant surgery. Optionally, heme oxygenase-1 (HO-1) can be providedto a patient in conjunction with administration of any or all of theproducts of heme degradation, e.g., carbon monoxide (CO), biliverdin,bilirubin, iron, and ferritin. Alternatively HO-1 can be provided to thepatient instead of providing any or all of the products of hemedegradation to the patient.

Use of Heme Oxygenase-1 and Products of Heme Degradation HemeOxygenase-1

HO-1 can be provided to a patient by inducing or expressing HO-1 in thepatient, or by administering exogenous HO-1 directly to the patient. Asused herein, the term “induce(d)” means to cause increased production ofa protein, e.g., HO-1 or ferritin, in the body of a patient, using thepatient's own endogenous (e.g., non-recombinant) gene that encodes theprotein.

HO-1 can be induced in a patient by any method known in the art. Forexample, production of HO-1 can be induced by hemin, by ironprotoporphyrin, or by cobalt protoporphyrin. A variety of non-hemeagents including heavy metals, cytokines, hormones, nitric oxide, COCl₂,endotoxin and heat shock are also strong inducers of HO-1 expression(Otterbein et al., Am. J. Physiol. Lung Cell Mol. Physiol.279:L1029-L1037, 2000; Choi et al., Am. J. Respir. Cell Mol. Biol.15:9-19, 1996; Maines, Annu. Rev. Pharmacol. Toxicol. 37:517-554, 1997;and Tenhunen et al., J. Lab. Clin. Med. 75:410-421, 1970). HO-1 is alsohighly induced by a variety of agents and conditions that createoxidative stress, including hydrogen peroxide, glutathione depletors, UVirradiation and hyperoxia (Choi et al., Am. J. Respir. Cell Mol. Biol.15: 9-19, 1996; Maines, Annu. Rev. Pharmacol. Toxicol. 37:517-554, 1997;and Keyse et al., Proc. Natl. Acad. Sci. USA 86:99-103, 1989). A“pharmaceutical composition comprising an inducer of HO-1” means apharmaceutical composition containing any agent capable of inducing HO-1in a patient, e.g., any of the agents described herein, e.g., hemin,iron protoporphyrin, and/or cobalt protoporphyrin.

The present invention contemplates that HO-1 can be expressed in apatient via gene transfer. As used herein, the term “express(ed)” meansto cause increased production of a protein, e.g., HO-1 or ferritin, inthe body of a patient using an exogenously administered gene (e.g., arecombinant gene). The HO-1 or ferritin is preferably of the samespecies (e.g., human, mouse, rat, etc.) as the patient, in order tominiinze any immune reaction. Expression could be driven by aconstitutive promoter (e.g., cytomegalovirus promoters) or atissue-specific promoter (e.g., milk whey promoter for mammary cells oralbumin promoter for liver cells). An appropriate gene therapy vector(e.g., retroviruses, adenoviruses, adeno-associated viruses (AAV), pox(e.g., vaccinia) viruses, human immunodeficiency virus (HIV), the minutevirus of mice, hepatitis B virus, influenza virus, Herpes SimplexVirus-1, and lentiviruses) encoding HO-1 or ferritin would beadministered to the patient orally, by inhalation, or by injection at alocation appropriate for treatment of a condition described herein.Particularly preferred is local administration directly to the site ofthe condition. Similarly, plasmid vectors encoding HO-1 or ferritin canbe administered, e.g., as naked DNA, in liposomes, or in microparticles.

Further, exogenous HO-1 protein can be directly administered to apatient by any method known in the art. Exogenous HO-1 can be directlyadministered in addition to, or as an alternative to the induction orexpression of HO-1 in the patient as described herein. The HO-1 proteincan be delivered to a patient, for example, in liposomes, and/or as afusion protein, e.g., as a TAT-fusion protein (see, e.g., Becker-Hapaket al., Methods 24, 247-256, 2001). In the context of surgicalprocedures such as transplantation, it is contemplated that HO-1 can beinduced and/or expressed in, and/or administered to donors, recipients,and/or the organ to be transplanted.

Heme Degradation Products

Additionally or alternatively, product(s) of heme degradation can beadministered to patients to treat the diseases or conditions describedherein. “Heme degradation products” include carbon monoxide, iron,biliverdin, bilirubin and (apo)ferritin. Any of the above can beprovided to patients, e.g., as an active ingredient in a pharmaceuticalcomposition or by other methods as described herein.

Biliverdin and Bilirubin

The terms “biliverdin” and “bilirubin” refer to the linear tetrapyrrolecompounds that are produced as a result of heme degradation.

Pharmaceutical compositions comprising biliverdin and/or bilirubin aretypically administered to patients in aqueous or solid forms. Biliverdinand bilirubin useful in the methods of the invention can be obtainedfrom any commercial source, e.g., any source that supplies biochemicalsfor medical or laboratory use. In the preparation, use, or storage ofbiliverdin and bilirubin, it is recommended that the compounds beexposed to as little light as possible.

The amount of biliverdin and/or bilirubin to be included inpharmaceutical compositions and to be administered to patients willdepend on absorption, distribution, inactivation, and excretion rates ofthe bilirubin and/or biliverdin, as well as other factors known to thoseof skill in the art. Effective amounts of biliverdin and/or bilirubinare amounts that are effective for treating a particular disease orcondition.

Effective amounts of biliverdin can fall within the range of about 1 to1000 micromoles/kg/day, e.g., at least 10 μmols/kg/day, e.g., at least10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, or 900 micromoles/kg/day. Preferred ranges include 10 to 500μmols/kg/day, 20 to 200 μmols/kg/day, and 25 to 100 μmols/kg/day.Because biliverdin is rapidly converted to bilirubin in the body (viabiliverdin reductase), the present invention contemplates that doses ofbiliverdin above 1000 micromoles/kg/day can be administered to patients.The entire dose of biliverdin can be administered as a single dose, inmultiple doses, e.g., several doses per day, or by constant infusion.

Effective amounts of bilirubin can be administered to a patient togenerate serum levels of bilirubin in a range of from about 1 to about300 μmols/L, e.g., at least about 10 to about 200 μmols/L, or about 50to about 100 pmols/L. To generate such serum levels, individual doses ofbilirubin can be administered, which can fall within the range of about1 to 1000 mg/kg, e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, or 900 mg/kg. Preferred rangesinclude 10 to 500 mg/kg, 20 to 200 mg/kg, and 25 to 150 mg/kg. Theentire dose of bilirubin can be administered as a single dose, inmultiple doses, e.g., several doses per day, or by constant infusion.

A skilled practitioner will appreciate that amounts of bilirubin and/orbiliverdin outside of these ranges can be used depending upon theapplication. Acute, sub-acute, and chronic administration ofpharmaceutical compositions comprising biliverdin and/or bilirubin arecontemplated by the present invention, depending upon, e.g., theseverity or persistence of the disease or condition in the patient. Thecompositions can be delivered to the patient for a time (includingindefinitely) sufficient to treat the condition and exert the intendedpharmacological or biological effect.

The present invention contemplates that biliverdin and/or bilirubin canbe bound to carriers. Such carriers include, for example, albumin orcyclodextrin. Binding of biliverdin and/or bilirubin to such a carrierscould increase the solubility of biliverdin and/or bilirubin, therebypreventing deposition of biliverdin and/or bilirubin in the tissues. Thepresent invention contemplates that it is possible to individuallyadminister unbound biliverdin and/or bilirubin and albumin to thepatient to produce the desired effect.

Alternatively or in addition, it is contemplated that biliverdinreductase can be induced, expressed, and/or administered to a patient insituations where it is deemed desirable to increase bilirubin levels inthe patient. The biliverdin reductase protein can be delivered to apatient, for example, in liposomes. Further, the present inventioncontemplates that increased levels of biliverdin reductase can begenerated in a patient via gene transfer. An appropriate gene therapyvector (e.g., plasmid, adenovirus, adeno-associated virus (AAV),lentivirus, or any of the other gene therapy vectors mentioned herein)that encodes biliverdin reductase, with the coding sequence operablylinked to an appropriate expression control sequence, would beadministered to the patient orally, via inhalation, or by injection at alocation appropriate for treatnent of a condition described herein. Inone embodiment of the present invention, a vector that encodesbiliverdin reductase is administered to an organ affected by a conditiondescribed herein and biliverdin is subsequently or simultaneouslyadministered to the organ, such that the biliverdin reductase breaksdown the biliverdin to produce bilirubin in the organ.

Iron and Ferritin

The release of free iron by the action of HO-1 on heme stimulates theinduction of apoferritin, which rapidly sequesters the iron to formferritin. The present invention includes inducing or expressing ferritinin a patient to treat inflammation or ischemia or cell proliferationassociated with various diseases or conditions in the patient. Ferritincan be induced in a patient by any method known in the art. For example,ferritin can be induced by administering iron dextran to the patient. Asanother example, ferritin levels in a patient can be increased byexposing the patient to ultraviolet radiation (Otterbein et al., Am. J.Physiol. Lung Cell Mol. Physiol. 279:L1029-L1037, 2000).

A “pharmaceutical composition comprising an inducer of ferritin” means apharmaceutical composition containing any agent capable of inducingferritin, e.g., heme, iron, and/or iron dextran, in a patient.Typically, a pharmaceutical composition comprising an inducer offerritin is administered to a patient in aqueous or solid form. Inducersof ferritin, e.g., iron or iron dextran, useful in the methods of theinvention can be obtained from any commercial source, e.g., a commercialsource that supplies chemicals for medical or laboratory use.

An effective amount of an inducer of ferritin, e.g., iron or irondextran, is an amount that is effective for treating a disease orcondition. Effective doses of iron dextran can be administered once orseveral times per day, and each dose can fall within the range of about1 to 1000 mg/kg, e.g., at least 2, 2.5, 5, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 800, or 900 mg/kg.Preferred ranges for iron dextran include 10 to 900 mg/kg, 100 to 800mg/kg, 300 to 700 mg/kg, or 400 to 600 mg/kg. Free iron can be deliveredto the patient, for example, as one or multiple doses of a commerciallyavailable iron supplement, e.g., a tablet containing iron.

Further, the present invention contemplates that increased levels offerritin, e.g., H-chain ferritin, can be generated in a patient via genetransfer. An appropriate gene therapy vector (as described herein) wouldbe administered to the patient orally or by injection or implantation ata location appropriate for treatment of a condition described herein.Further, exogenous ferritin can be directly administered to a patient byany method known in the art. Exogenous ferritin can be directlyadministered in addition to, or as an alternative to the induction orexpression of ferritin in the patient as described herein. The ferritinprotein can be delivered to a patient, for example, in liposomes, and/oras a fusion protein, e.g., as a TAT-fusion protein (see, e.g.,Becker-Hapak et al., Methods 24:247-256, 2001).

Alternatively or in addition, it is contemplated that other iron-bindingmolecules can be administered to the patient to create or enhance thedesired effect, e.g., to reduce free iron levels. As one example, thepresent invention contemplates that apoferritin can be administered to apatient, as well as any type of iron chelator, e.g., desferoxamine (DFO)or salicylaldehyde isonicotinoyl hydrazone (SIH) (see, e.g., Blaha etal., Blood 91(11):4368-4372, 1998) to create or enhance the desiredeffect.

Effective doses of DFO can be administered once or several times perday, and each dose can fall within the range of from about 0.1 to 1000mg/kg, e.g., at least 2, 2.5., 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 250, 300, 400, 500, 600, 700, 800, or 900 mg/kg. Preferredranges for DFO include 0.5 to 800 mg/kg, 1 to 600 mg/kg, 2 to 400 mg/kg,or 2.5 to 250 mg/kg.

Effective doses of SIH can be administered once or several times perday, and each dose can fall within the range of from about 0.02 to 100mmol/kg, e.g., 0.02 to 50 mmol/kg, or 0.2 to 20 mmol/kg.

Effective doses of apoferritin can be administered once or several timesper day, and each dose can fall within the range of about 1 to 1000mg/kg, e.g., at least 2, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 250, 300, 400, 500, 600, 700, 800, or 900 mg/kg. Preferredranges include 10 to 500 mg/kg, 20 to 200 mg/kg, and 25 to 150 mg/kg.

The skilled practitioner will recognize that any of the above, e.g.,iron chelators, e.g., DFO or SIH, iron dextran, and apoferritin, can beadministered as a single dose, in multiple doses, e.g., several dosesper day, or by constant infusion. Further any of the above can beadministered continuously, and for as long as necessary to produce thedesired effect. Further, the skilled practitioner will recognize thatany of the above can be administered in amounts outside the rangesgiven, depending upon the application.

Carbon Monoxide

The term “carbon monoxide” (or “CO”) as used herein describes molecularcarbon monoxide in its gaseous state, compressed into liquid form, ordissolved in aqueous solution. An effective amount of carbon monoxidefor use in the present invention is an amount that is effective fortreating a disease or condition. For gases, effective amounts of carbonmonoxide generally fall within the range of about 0.0000001% to about0.3% by weight, e.g., 0.0001% to about 0.25% by weight, preferably atleast about 0.001%, e.g., 0.005%, 0.010%, 0.02%, 0.025%, 0.03%, 0.04%,0.05%, 0.06%, 0.08%, 0.10%, 0.15%, 0.20%, 0.22%, or 0.24% by weight ofcarbon monoxide. For liquid solutions of CO, effective amounts generallyfall within the range of about 0.0001 to about 0.0044 g CO/100 g liquid,e.g., 0.0001, 0.0002, 0.0004, 0.0006, 0.0008, 0.0010, 0.0013, 0.0014,0.0015, 0.0016, 0.0018, 0.0020, 0.0021, 0.0022, 0.0024, 0.0026, 0.0028,0.0030, 0.0032, 0.0035, 0.0037, 0.0040, or 0.0042 g CO/100 g aqueoussolution. A skilled practitioner will appreciate that amounts outside ofthese ranges can be used depending upon the application.

A carbon monoxide composition can be a gaseous carbon monoxidecomposition. Compressed or pressurized gas useful in the methods of theinvention can be obtained from any commercial source, and in any type ofvessel appropriate for storing compressed gas. For example, compressedor pressurized gases can be obtained from any source that suppliescompressed gases, such as oxygen, for medical use. The pressurized gasincluding carbon monoxide used in the methods of the present inventioncan be provided such that all gases of the desired final composition(e.g., CO, He, NO, CO₂, O₂, N₂) are in the same vessel. Optionally, themethods of the present invention can be performed using multiple vesselscontaining individual gases. For example, a single vessel can beprovided that contains carbon monoxide, with or without other gases, thecontents of which can be optionally mixed with the contents of othervessels, e.g., vessels containing oxygen, nitrogen, carbon dioxide,compressed air, or any other suitable gas or mixtures thereof.

Gaseous carbon monoxide compositions administered to a patient accordingto the present invention typically contain 0% to about 79% by weightnitrogen, about 21% to about 100% by weight oxygen and about 0.0000001%to about 0.3% by weight (corresponding to about 1 ppb or 0.001 ppm toabout 3,000 ppm) carbon monoxide. Preferably, the amount of nitrogen inthe gaseous composition is about 79% by weight, the amount of oxygen isabout 21% by weight and the amount of carbon monoxide is about 0.0001%to about 0.25% by weight, preferably at least about 0.001%, e.g.,0.005%, 0.010%, 0.02%, 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0.10%,0.15%, 0.20%, 0.22%, or 0.24% by weight of carbon monoxide. It is notedthat gaseous carbon monoxide compositions having concentrations ofcarbon monoxide greater than 0.3% (such as 1% or greater) can be usedfor short periods (e.g., one or a few breaths), depending upon theapplication.

A gaseous carbon monoxide composition can be used to create anatmosphere that comprises carbon monoxide gas. An atmosphere thatincludes appropriate levels of carbon monoxide gas can be created, forexample, by providing a vessel containing a pressurized gas comprisingcarbon monoxide gas, and releasing the pressurized gas from the vesselinto a chamber or space to form an atmosphere that includes the carbonmonoxide gas inside the chamber or space. Alternatively, the gases canbe released into an apparatus that culminates in a breathing mask orbreathing tube, thereby creating an atmosphere comprising carbonmonoxide gas in the breathing mask or breathing tube, ensuring thepatient is the only person in the room exposed to significant levels ofcarbon monoxide.

Carbon monoxide levels in an atmosphere can be measured or monitoredusing any method known in the art. Such methods include electrochemicaldetection, gas chromatography, radioisotope counting, infraredabsorption, colorimetry, and electrochemical methods based on selectivemembranes (see, e.g., Sunderman et al., Clin. Chem. 28:2026-2032, 1982;Ingi et al., Neuron 16:835-842, 1996). Sub-parts per million carbonmonoxide levels can be detected by, e.g., gas chromatography andradioisotope counting. Further, it is known in the art that carbonmonoxide levels in the sub-ppm range can be measured in biologicaltissue by a midinfrared gas sensor (see, e.g., Morimoto et al., Am. J.Physiol. Heart. Circ. Physiol 280:H482-H488, 2001). Carbon monoxidesensors and gas detection devices are widely available from manycommercial sources.

A pharmaceutical composition comprising carbon monoxide can also be aliquid composition. A liquid can be made into a pharmaceuticalcomposition comprising carbon monoxide by any method known in the artfor causing gases to become dissolved in liquids. For example, theliquid can be placed in a so-called “CO₂ incubator” and exposed to acontinuous flow of carbon monoxide, preferably balanced with carbondioxide, until a desired concentration of carbon monoxide is reached inthe liquid. As another example, carbon monoxide gas can be “bubbled”directly into the liquid until the desired concentration of carbonmonoxide in the liquid is reached. The amount of carbon monoxide thatcan be dissolved in a given aqueous solution increases with decreasingtemperature. As still another example, an appropriate liquid can bepassed through tubing that allows gas diffusion, where the tubing runsthrough an atmosphere comprising carbon monoxide (e.g., utilizing adevice such as an extracorporeal membrane oxygenator). The carbonmonoxide diffuses into the liquid to create a liquid carbon monoxidecomposition.

The liquid can be any liquid known to those of skill in the art to besuitable for administration to patients (see, for example, OxfordTextbook of Surgery, Morris and Malt, Eds., Oxford University Press,1994). In general, the liquid will be an aqueous solution. Examples ofsolutions include Phosphate Buffered Saline (PBS), Celsior™, Perfadex™,Collins solution, citrate solution, and University of Wisconsin (UW)solution (Oxford Textbook of Surgery, Morris and Malt, Eds., OxfordUniversity Press, 1994).

The present invention contemplates that compounds that release CO intothe body after administration of the compound (e.g., CO-releasingcompounds, e.g., photoactivatable CO-releasing compounds), e.g.,dimanganese decacarbonyl, tricarbonyldichlororuthenium (II) dimer, andmethylene chloride (e.g., at a dose of between 400 to 600 mg/kg, e.g.,about 500 mg/kg), can also be used in the methods of the presentinvention, as can carboxyhemoglobin and CO-donating hemoglobinsubstitutes. Agents capable of delivering doses of CO gas or liquid canalso be utilized (e.g., CO releasing gums, creams, ointments or patches)

Any suitable liquid can be saturated to a set concentration of carbonmonoxide via gas diffusers. Alternatively, pre-made solutions that havebeen quality controlled to contain set levels of carbon monoxide can beused. Accurate control of dose can be achieved via measurements with agas permeable, liquid impermeable membrane connected to a carbonmonoxide analyzer. Solutions can be saturated to desired effectiveconcentrations and maintained at these levels.

A patient can be treated with a carbon monoxide composition by anymethod known in the art of administering gases and/or liquids topatients. The present invention contemplates the systemic administrationof liquid or gaseous carbon monoxide compositions to patients (e.g., byinhalation and/or ingestion), and the topical administration of thecompositions to the patient's organs, e.g., the gastrointestinal tract.

Gaseous carbon monoxide compositions are typically administered byinhalation through the mouth or nasal passages to the lungs, where thecarbon monoxide can exert its effect directly or be readily absorbedinto the patient's bloodstream. The concentration of active compound(CO) utilized in the therapeutic gaseous composition will depend onabsorption, distribution, inactivation, and excretion (generally,through respiration) rates of the carbon monoxide as well as otherfactors known to those of skill in the art. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and that the concentration rangesset forth herein are exemplary only and are not intended to limit thescope or practice of the claimed invention. Acute, sub-acute and chronicadministration of carbon monoxide are contemplated by the presentinvention, depending upon, e.g., the severity or persistence of diseaseor condition in the patient. Carbon monoxide can be delivered to thepatient for a time (including indefinitely) sufficient to treat thecondition and exert the intended pharmacological or biological effect.

Examples of methods and devices that can be utilized to administergaseous pharmaceutical compositions comprising carbon monoxide topatients include ventilators, face masks and tents, portable inhalers,intravenous artificial lungs (see, e.g., Hattler et al., Artif. Organs18(11):806-812, 1994; and Golob et al., ASAIO J., 47(5):432-437, 2001),and normobaric chambers, as described in further detail below.

The present invention further contemplates that aqueous solutionscomprising carbon monoxide can be created for systemic delivery to apatient, e.g., by oral delivery to a patient.

Alternatively or in addition, carbon monoxide compositions can beapplied directly to the organs of a patient. For example, carbonmonoxide compositions can be applied to the interior and/or exterior ofthe entire gastrointestinal tract, or to any portion thereof, by anymethod known in the art for insufflating gases into a patient. Forexample, gases, e.g., carbon dioxide, are often insufflated into thegastrointestinal tract and the abdominal cavity of patients tofacilitate examination during endoscopic and laproscopic procedures,respectively (see, e.g., Oxford Textbook of Surgery, Morris and Malt,Eds., Oxford University Press, 1994). The skilled practitioner willappreciate that similar procedures could be used to administer carbonmonoxide compositions directly to the gastrointestinal tract of apatient.

Aqueous carbon monoxide compositions can also be administered directlyto the organs of a patient. Aqueous forms of the compositions can beadministered by any method known in the art for administering liquids topatients. For example, the aqueous form can be administered orally,e.g., by causing the patient to ingest an encapsulated or unencapsulateddose of the aqueous carbon monoxide composition. As another example,liquids, e.g., saline solutions, can be injected into thegastrointestinal tract and the abdominal cavity of patients duringendoscopic and laparoscopic procedures, respectively. The skilledpractitioner will appreciate that similar procedures could be used toadmister liquid carbon monoxide compositions directly to the organs of apatient.

Combination Therapy

The present invention contemplates that any of the treatments describedherein, e.g., induction/expression/administration of HO-1 and/orferritin, and the administration of CO, bilirubin, and/or biliverdin,can be used individually or in any combination in surgical proceduresand to treat the disorders or conditions described herein. Further, thepresent invention contemplates that in any treatment regimen using anycombination of the herein-described treatments, the treatments can beadministered simultaneously on a single or multiple occasions, and/orindividually at varying points in time, e.g., at different phases of adisease or condition. For example, a patient can receive both bilirubinand iron, or both of those plus CO, or bilirubin plus ferritin, or twoor more inducers of HO-1.

Treatment of Patients with Pharmaceutical Compositions of the PresentInvention

A patient can be treated with pharmaceutical compositions describedherein by any method known in the art of administering liquids, solids,and/or gases to a patient.

Systemic Deliver of Pharmaceutical Compositions

Aqueous and Solid Pharmaceutical Compositions

The present invention contemplates that aqueous pharmaceuticalcompositions can be created for systemic delivery to a patient byinjection into the body, e.g., intravenously, intra-arterially,intraperitoneally, and/or subcutaneously. Aqueous pharmaceuticalcompositions can also be prepared for oral delivery, e.g., inencapsulated or unencapsulated form, to be absorbed in any portion ofthe gastrointestinal tract, e.g., the stomach or small intestine.Similarly, solid pharmaceutical compositions can be created for systemicdelivery to a patient, e.g., in the form of a powder or an ingestiblecapsule.

Aqueous and solid pharmaceutical compositions typically include theactive ingredient and a pharmaceutically acceptable carrier. As usedherein the language “pharmaceutically acceptable carrier” includessolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Supplementary activecompounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oraland/or rectal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; buffers such as acetates, citrates or phosphates and agentsfor the adjustment of tonicity such as sodium chloride or dextrose. pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, isotonic agents,e.g., sugars, polyalcohols such as mannitol or sorbitol, or sodiumchloride can be included in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin. Microbeads, microspheres, or any otherphysiologicially-acceptable methods, e.g., encapsulation, can be used todelay release or absorption of the active ingredients.

Sterile injectable solutions can be prepared by incorporating the activeingredient in the required amount in an appropriate solvent with one ora combination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying, and freeze-drying that yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions, which can be aqueous or solid, generally include aninert diluent or an edible carrier. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules, e.g., gelatincapsules. Pharmaceutically compatible binding agents and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose; a disintegrating agent such as alginic acid,Primogel™, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

The active ingredients can be prepared with carriers that will protectthe compound against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations will be apparent to those skilled in the art. Thematerials can also be obtained commercially from Alza Corporation andNova Pharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies specific for viralantigens) can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetemmined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the EDSO (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

Gaseous Pharmaceutical Compositions

Gaseous pharmaceutical compositions, e.g., pharmaceutical compositionscontaining carbon monoxide, can be delivered systemically to a patientby inhalation-through the mouth or nasal passages to the lungs. Thefollowing methods and apparatus for administering carbon monoxidecompositions are illustrative of useful systemic delivery methods forthe gaseous pharmaceutical compositions described herein.

Ventilators

Medical grade carbon monoxide (concentrations can vary) can be purchasedmixed with air or another oxygen-containing gas in a standard tank ofcompressed gas (e.g., 21% O₂, 79% N₂). It is non-reactive, and theconcentrations that are required for the methods of the presentinvention are well below the combustible range (10% in air). In ahospital setting, the gas presumably will be delivered to the bedsidewhere it will be mixed with house air in a blender to a desiredconcentration in ppm (parts per million). The patient will inhale thegas mixture through a ventilator, which will be set to a flow rate basedon patient comfort and needs. This is determined by pulmonary graphics(i.e., respiratory rate, tidal volumes etc.). Fail-safe mechanism(s) toprevent the patient from unnecessarily receiving greater than desiredamounts of carbon monoxide can be designed into the delivery system. Thepatient's carbon monoxide level can be monitored by studying (1)carboxyhemoglobin (COHb), which can be measured in venous blood, and (2)exhaled carbon monoxide collected from a side port of the ventilator.Carbon monoxide exposure can be adjusted based upon the patient's healthstatus and on the basis of the markers. If necessary, carbon monoxidecan be washed out of the patient by switching to 100% O₂ inhalation.Carbon monoxide is not metabolized; thus, whatever is inhaled willultimately be exhaled except for a very small percentage that isconverted to CO₂. Carbon monoxide can also be mixed with any level of O₂to provide therapeutic delivery of carbon monoxide without consequentialhypoxic conditions.

Face Mask and Tent

A carbon monoxide containing gas mixture is prepared as above to allowpassive inhalation by the patient using a facemask or tent. Theconcentration inhaled can be changed and can be washed out by simplyswitching over to 100% O₂. Monitoring of carbon monoxide levels wouldoccur at or near the mask or tent with a fail-safe mechanism that wouldprevent too high of a concentration of carbon monoxide from beinginhaled.

Portable Inhaler

Compressed carbon monoxide can be packaged into a portable inhalerdevice and inhaled in a metered dose, for example, to permitinternittent treatment of a recipient who is not in a hospital setting.Different concentrations of carbon monoxide could be packaged in thecontainers. The device could be as simple as a small tank (e.g., under 5kg) of appropriately diluted CO with an on-off valve and a tube fromwhich the patient takes a whiff of CO according to a standard regimen oras needed.

Intravenous Artificial Lung

An artificial lung (a catheter device for gas exchange in the blood)designed for O₂ delivery and CO₂ removal can be used for carbon monoxidedelivery. The catheter, when implanted, resides in one of the largeveins and would be able to deliver carbon monoxide at givenconcentrations either for systemic delivery or at a local site. Thedelivery can be a local delivery of a high concentration of carbonmonoxide for a short period of time at the site of the procedure, e.g.,in proximity to the small intestine (this high concentration wouldrapidly be diluted out in the bloodstream), or a relatively longerexposure to a lower concentration of carbon monoxide (see, e.g., Hattleret al., Artif. Organs 18(11):806-812, 1994; and Golob et al., ASAIO J.,47(5):432-437, 2001).

Normobaric Chamber

In certain instances, it would be desirable to expose the whole patientto carbon monoxide. The patient would be inside an airtight chamber thatwould be flooded with carbon monoxide (at a level that does not endangerthe patient, or at a level that poses an acceptable risk, or fornon-human donors or brain-dead donors, at any desired level) without therisk of bystanders being exposed. Upon completion of the exposure, thechamber could be flushed with air (e.g., 21% O₂, 79% N₂) and samplescould be analyzed by carbon monoxide analyzers to ensure no carbonmonoxide remains before allowing the patient to exit the exposuresystem.

Topical Delivery of Pharmaceutical Compositions

Alternatively or in addition, pharmaceutical compositions can be applieddirectly to an organ, tissue, or area of the patient's body to betreated.

Aqueous and Solid Pharmaceutical Compositions

Aqueous and solid pharmaceutical compositions can also be directlyapplied to an organ of a patient, or to an area of the patient targetedfor treatment, by any method known in the art for administering liquidsor solids to patients. For example, an aqueous or solid composition canbe administered orally, e.g., by causing the patient to ingest anencapsulated or unencapsulated dose of the aqueous or solidpharmaceutical composition, to treat the interior of thegastrointestinal tract or any portion thereof. Further, liquids, e.g.,saline solutions, are often injected into the gastrointestinal tract andthe abdominal cavity of patients during endoscopic and laparoscopicprocedures, respectively. The skilled practitioner will appreciate thatsimilar procedures could be used to administer aqueous pharmaceuticalcompositions directly to an organ or e.g., in the vicinity of an organto be treated, to thereby expose the organ in situ to an aqueouspharmaceutical composition.

In the context of transplantation, in situ exposures can be performed byany method known in the art, e.g., by in situ flushing of the organ witha liquid pharmaceutical composition prior to removal from the donor (seeOxford Textbook of Surgery, Morris and Malt, Eds., Oxford UniversityPress, 1994). Such exposures are described in further detail below.

Gaseous Pharmaceutical Compositions

A gaseous pharmaceutical composition can be directly applied to an organof a patient, or to an area of the patient targeted for treatment, byany method known in the art for insufflating gases into a patient. Forexample, gases, e.g., carbon dioxide, are often insufflated into thegastrointestinal tract and the abdominal cavity of patients tofacilitate examination during endoscopic and laparoscopic procedures,respectively (see, e.g., Oxford Textbook of Surgery, Morris and Malt,Eds., Oxford University Press, 1994). The skilled practitioner willappreciate that similar procedures could be used to administer gaseouspharmaceutical compositions directly to the interior of thegastrointestinal tract, or any portion thereof. Further, the skilledpractitioner will appreciate that gaseous pharmaceutical compositionscan be insufflated into the abdominal cavity of patients, e.g., in thevicinity of an organ to be treated, to thereby expose the organ in situto a gaseous pharmaceutical composition.

Surgical Procedures: Transplantation

The present invention contemplates the use of the methods describedherein to treat patients who undergo transplantation. The methods can beused to treat donors, recipients and/or the organ at any step of theorgan harvesting, storage, and transplant process. For example, an organcan be harvested from a donor, treated with a pharmaceutical compositionex vivo in accordance with the present invention, and transplanted intoa recipient. Alternatively or in addition, the organ can be treated insitu, while still in the donor (by treatment of the donor or by treatingthe organ). Optionally, a pharmaceutical composition can be administeredto the recipient prior to, during, and/or after the surgery, e.g., afterthe organ is reperfused with the recipient's blood. The composition canbe administered to the donor prior to or during the process ofharvesting the organ from the donor.

The terms “transplantation” is used throughout the specification as ageneral term to describe the process of transferring an organ to apatient. The term “transplantation” is defined in the art as thetransfer of living tissues or cells from a donor to a recipient, withthe intention of maintaining the functional integrity of thetransplanted tissue or cells in the recipient (see, e.g., The MerckManual, Berkow, Fletcher, and Beers, Eds., Merck Research Laboratories,Rahway, N.J., 1992). The term includes all categories of transplantsknown in the art. Transplants are categorized by site and geneticrelationship between donor and recipient. The term includes, e.g.,autotransplantation (removal and transfer of cells or tissue from onelocation on a patient to the same or another location on the samepatient), allotransplantation (transplantation between members of thesame species), and xenotransplantation (transplantations between membersof different species).

The term “donor” or “donor patient” as used herein refers to an animal(human or non-human) from whom an organ or tissue can be obtained forthe purposes of storage and/or transplantation to a recipient patient.The term “recipient” or “recipient patient” refers to an animal (humanor non-human) into which an organ or tissue can be transferred.

The terms “organ rejection,” “transplant rejection,” or “rejection” areart-recognized, and are used throughout the specification as a generalterm to describe the process of rejection of an organ, tissues, or cellsin a recipient. Included within the definition are, for example, threemain patterns of rejection that are usually identified in clinicalpractice: hyperacute rejection, acute rejection, and chronic rejection(see, e.g., Oxford Textbook of Surgery, Morris and Malt, Eds., OxfordUniversity Press, 1994).

The term “organ(s)” is used throughout the specification as a generalterm to describe any anatomical part or member having a specificfunction in the animal. Further included within the meaning of this'term are substantial portions of organs, e.g., cohesive tissues obtainedfrom an organ. Further still, included within the meaning of this termare portions of an organ as small as one cell of the organ. Such organsinclude but are not limited to kidney; liver; heart; intestine, e.g.,large or small intestine; pancreas, e.g., islets; and lungs. Furtherincluded in this definition are bones, skin, and blood vessels.

Ex vivo exposure of an organ to a pharmaceutical composition can occurby exposing the organ to an atmosphere comprising a gaseouspharmaceutical composition, to a liquid pharmaceutical composition,e.g., a liquid perfusate, storage solution, or wash solution containingthe pharmaceutical composition, or to both.

For example, in the context of exposing an organ to a gaseouspharmaceutical composition comprising carbon monoxide, the exposure canbe performed in any chamber or area suitable for creating an atmospherethat includes appropriate levels of carbon monoxide gas. Such chambersinclude, for example, incubators and chambers built for the purpose ofaccommodating an organ in a preservation solution. An appropriatechamber can be a chamber wherein only the gases fed into the chamber arepresent in the internal atmosphere, such that the concentration ofcarbon monoxide can be established and maintained at a givenconcentration and purity, e.g., where the chamber is airtight. Forexample, a CO₂ incubator can be used to expose an organ to a carbonmonoxide composition, wherein carbon monoxide gas is supplied in acontinuous flow from a vessel that contains the gas.

As another example, in the context of exposing an organ to an aqueouspharmaceutical composition, the exposure can be performed in any chamberor space having sufficient volume for submerging the organ, completelyor partially, in an aqueous pharmaceutical composition. As yet anotherexample, the organ can be exposed by placing the organ in any suitablecontainer, and causing a liquid pharmaceutical composition to “washover” the organ, such that the organ is exposed to a continuous flow ofthe composition.

As another option, the organ can be perfused with an aqueouspharmaceutical composition. The term “perfusion” is an art-recognizedterm, and relates to the passage of a liquid, e.g., an aqueouspharmaceutical composition, through the blood vessels of the organ.Methods for perfusing organs ex vivo and in situ are well known in theart. An organ can be perfused with an aqueous pharmaceutical compositionex vivo, for example, by continuous hypothermic machine perfusion (seeOxford Textbook of Surgery, Morris and Malt, Eds., Oxford UniversityPress, 1994). The aqueous pharmaceutical solution can be allowed toremain in the vasculature for a given length of time. Optionally, in insitu or ex vivo perfusions, the organ can be perfused with a washsolution, e.g., UW solution without a pharmaceutical composition, priorto perfusion with the aqueous pharmaceutical composition to remove thedonor's blood from the organ. Such a process could be advantageous, forexample, when using pharmaceutical compositions comprising carbonmonoxide, to avoid competition for carbon monoxide by the donor'shemoglobin. As another option, the wash solution itself can be apharmaceutical composition, e.g., a pharmaceutical compositioncomprising carbon monoxide.

As yet another example, in the context of pharmaceutical compositionscomprising carbon monoxide, the organ can be placed, e.g., submerged, ina medium or solution that does not include carbon monoxide, and placedin a chamber such that the medium or solution can be made into a carbonmonoxide composition via exposure to a carbon monoxide-containingatmosphere as described herein. As still another example, the organ canbe submerged in a liquid that does not include carbon monoxide, andcarbon monoxide can be “bubbled” into the liquid.

An organ can be harvested from a donor, and transplanted by any methodsknown to those of skill in the art (see, for example, Oxford Textbook ofSurgery, Morris and Malt, Eds., Oxford University Press, 1994). Theskilled practitioner will recognize that methods for transplantingand/or harvesting organs for transplantation can vary depending uponmany circumstances, such as the age of the donor/recipient or the natureof the organ being transplanted.

The present invention contemplates that any or all of the above methodsfor exposing an organ to a pharmaceutical composition, e.g., washing,submerging, or perfusing, can be used in a given procedure, e.g., usedin a single transplantation procedure.

Surgical Procedures: Balloon Angioplasty and Surgically-Induced IntimalHyperplasia

The present invention contemplates the use of the methods describedherein to treat patients who undergo balloon angioplasty, or areotherwise at risk for intimal hyperplasia, e.g., due to vascularsurgery. Intimal hyperplasia from vascular injury subsequent toprocedures such as angioplasty, bypass surgery or organ transplantationcontinues to limit the success of these therapeutic interventions. Theterm “intimal hyperplasia” is an art-recognized term and is used hereinto refer to the proliferation of cells, e.g., smooth muscle cells and/ormyofibroblasts, within the intima. The skilled practitioner willappreciate that intimal hyperplasia can be caused by any number offactors, e.g., mechanical, chemical and/or immunological damage to theintima. Intimal hyperplasia can often be observed in patients, forexample, following balloon angioplasty or vascular surgery, e.g.,vascular surgery involving vein grafts. The term “angioplasty” is anart-recognized term and refers to any procedure involving the remodelingof an artery. Such procedures include, e.g., angioplasty using balloons(“balloon angioplasty”), lasers (“laser angioplasty”), and any othermode for performing angioplasty, e.g., using other suitable instruments,such as a microfabricated probe.

Individuals considered at risk for developing intimal hyperplasia maybenefit particularly from the invention, primarily because prophylactictreatment can begin before there is any evidence of intimal hyperplasia.Individuals “at risk” include, e.g., patients that have or will undergoangioplasty, e.g., balloon angioplasty, or patients that have or willhave any type of mechanical, chemical and/or immunological damage to theintima.

A patient can be treated according to the methods of the presentinvention before, during and/or after the surgical procedure orangioplasty. Further, if desired, vein(s) can be exposed to thepharmaceutical compositions described herein in situ and/or ex vivo, asdescribed herein in the context of organ transplants. The vein can beexposed to a gaseous pharmaceutical composition, and/or to a liquidpharmaceutical composition, e.g., a liquid perfusate, storage solution,or wash solution having the active ingredient dissolved therein. Forexample, a liquid pharmaceutical composition can be instilled into anarterial segment, e.g., by retrograde perfusion, and can be allowed toremain in the segment for a given length of time.

Disorders and Conditions

The methods of the present invention can be used to treat one or more ofthe following inflammatory, respiratory, cardiovascular, renal,hepatobiliary, reproductive, and gastrointestinal disorders; shock; orcellular proliferative and/or differentiative disorders, or to reducethe effects of ischemia, or aid in wound healing.

Respiratory Disorders

Examples of respiratory conditions include, but are not limited to,asthma; Acute Respiratory Distress Syndrome (ARDS), e.g., ARDS caused byperitonitis, pneumonia (bacterial or viral), or trauma; idiopathicpulmonary diseases; interstitial lung diseases, e.g., InterstitialPulmonary Fibrosis (IPF); pulmonary emboli; Chronic ObstructivePulmonary Disease (COPD); emphysema; bronchitis; cystic fibrosis; lungcancer of any type; lung injury, e.g., hyperoxic lung injury, PrimaryPulmonary Hypertension (PPH); secondary pulmonary hypertension; andsleep-related disorders, e.g., sleep apnea.

Cardiovascular Disorders

Cardiovascular disorders include disorders involving the cardiovascularsystem, e.g., the heart, the blood vessels, and/or the blood. Acardiovascular disorder can be caused, for example, by an imbalance inarterial pressure, a malfunction of the heart, or an occlusion of ablood vessel, e.g., by a thrombus. Examples of such disorders includecongestive heart failure, peripheral vascular disease, pulmonaryvascular thrombotic diseases such as pulmonary embolism, stroke,ischemia-reperfusion (I/R) injury to the heart, atherosclerosis, andheart attacks.

Renal Disorders

Disorders involving the kidney include, but are not limited to,pathologies of glomerular injury such as in situ immune complexdeposition and cell-mediated immunity in glomerulonephritis; damagecaused by activation of alternative complement pathway, epithelial cellinjury; pathologies involving mediators of glomerular injury includingcellular and soluble mediators; acute glomerulonephritis, such as acuteproliferative glomerulonephritis, e.g., poststreptococcalglomerulonephritis and nonstreptococcal acute glomerulonephritis,rapidly progressive glomerulonephritis, nephrotic syndrome, membranousglomerulonephritis (membranous nephropathy), minimal change disease(lipoid nephrosis), focal segmental glomerulosclerosis,membranoproliferative glomerulonephritis, IgA nephropathy (Bergerdisease), focal proliferative and necrotizing glomerulonephritis (focalglomerulonephritis) and chronic glomerulonephritis. Disorders of thekidney also include infections of the genitourinary tract.

Hepatobiliary Disorders

Disorders involving the liver include, but are not limited to, cirrhosisand infectious disorders such as viral hepatitis, including hepatitisA-E viral infection and infection by other hepatitis viruses,clinicopathologic syndromes, acute viral hepatitis, chronic viralhepatitis, and fulminant hepatitis; and drug- and toxin-induced liverdisease, such as alcoholic liver disease.

Gastrointestinal Disorders

Gastrointestinal disorders include, but are not limited to, ileus (ofany portion of the gastrointestinal tract, e.g., the large or smallintestine); inflammatory bowel disease, e.g., specific inflammatorybowel disease, e.g., infective specific inflammatory bowel disease,e.g., amoebic or bacillary dysentery, schistosomiasis, campylobacterenterocolitis, yersinia enterocolitis, or enterobius vermicularis;non-infective specific inflammatory bowel disease, e.g., radiationenterocolitis, ischaemic colitis, or eosinophilic gastroenteritis;non-specific bowel disease, e.g., ulcerative colitis, indeterminatecolitis, and Crohn's disease; necrotizing enterocolitis (NEC); andpancreatitis.

Cellular Proliferative and/or Differentiative Disorders

Examples of cellular proliferative and/or differentiative disordersinclude, but are not limited to, cancer, e.g., carcinoma, sarcoma,metastatic disorders or hematopoietic neoplastic disorders, e.g.,leukemias. A metastatic tumor can arise from a multitude of primarytumor types, including but not limited to those of prostate, colon,lung, breast and liver origin. The term “cancer” refers to cells havingthe capacity for autonomous growth. Examples of such cells include cellshaving an abnormal state or condition characterized by rapidlyproliferating cell growth. The term is meant to include all types ofcancerous growths, e.g., tumors, or oncogenic processes, or metastatictissues. Also included are malignancies of the various organ systems,such as lung, breast, thyroid, lymphoid, gastrointestinal, andgenito-urinary tract, as well as adenocarcinomas which includemalignancies such as most colon cancers, renal-cell carcinoma, prostatecancer and/or testicular tumors, non-small cell carcinoma of the lung,cancer of the small intestine and cancer of the esophagus. The term“carcinoma” is art recognized and refers to malignancies of epithelialor endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. Exemplary carcinomas includethose forming from tissue of the cervix, lung, prostate, breast, headand neck, colon and ovary. The term also includes carcinosarcomas, e.g.,which include malignant tumors composed of carcinomatous and sarcomatoustissues. An “adenocarcinoma” refers to a carcinoma derived fromglandular tissue or in which the tumor cells form recognizable glandularstructures.

The term “sarcoma” is art recognized and refers to malignant tumors ofmesenchymal derivation. The term “hematopoietic neoplastic disorders”includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin. A hematopoietic neoplastic disorder can arise frommyeloid, lymphoid or erythroid lineages, or precursor cells thereof.

Cancers which can be treated using the present compositions and methodsinclude, for example, stomach, colon, rectal, liver, pancreatic, lung,breast, cervix uteri, corpus uteri, ovary, prostate, testis, bladder,renal, brain/central nervous system, head and neck, throat, Hodgkinsdisease, non-Hodgkins leukemia, skin melanoma, various sarcomas, smallcell lung cancer, choriocarcinoma, mouth/pharynx, esophagus, larynx,melanoma, and kidney and lymphoma, among others.

Neurological Disorders

The methods of the present invention can also be used to treatneurological disorders. Neurological disorders include, but are notlimited to disorders involving the brain, e.g., degenerative diseasesaffecting the cerebral cortex, including Alzheimer's disease, anddegenerative diseases of basal ganglia and brain stem, includingParkinsonism and idiopathic Parkinson's disease (paralysis agitans).Further, the methods can be used to treat pain disorders. Examples ofpain disorders include, but are not limited to, pain response elicitedduring various forms of tissue injury, e.g., inflammation, infection,and ischemia, usually referred to as hyperalgesia (described in, forexample, Fields, Pain, New York:McGraw-Hill, 1987); pain associated withmusculoskeletal disorders, e.g., joint pain; tooth pain; headaches; painassociated with surgery; pain related to irritable bowel syndrome; orchest pain. Also included in this category are seizure disorders, e.g.,epilepsy.

Inflammatory Disorders

The methods of the present invention can be used to treat inflammatorydisorders. The terms “inflammatory disorder(s)” and “inflammation” areused to describe the fundamental pathological process consisting of adynamic complex of reactions (which can be recognized based on cytologicand histologic studies) that occur in the affected blood vessels andadjacent tissues in response to an injury or abnormal stimulation causedby a physical, chemical or biologic agent, including the local reactionsand resulting morphologic changes, the destruction or removal of theinjurious material, and the responses that lead to repair and healing.Inflammation is characterized in some instances by the infiltration ofcells immune cells such as monocytes/macrophages, natural killer cells,and/or lymphocytes (e.g., B and T lymphocytes) into the area of tissue.In addition, inflamed tissue may contain cytokines and chemokines thatare produced by the cells that have infiltrated into the area. Often,inflammation is accompanied by thrombosis, including both coagulationand platelet aggregation. The term inflammation includes various typesof inflammation such as acute, chronic, allergic (including conditionsinvolving mast cells), alterative (degenerative), atrophic, catarrhal(most frequently in the respiratory tract), croupous, fibrinopurulent,fibrinous, immune, hyperplastic or proliferative, subacute, serous andserofibrinous. Inflammation localized in the gastrointestinal tract, orany portion thereof, kidneys, liver, heart, skin, spleen, brain, kidney,pulmonary tract, and the lungs is favorably treated by the methods ofthe present invention. Inflammation associated with shock, e.g., septicshock, hemorrhagic shock caused by any type of trauma, and anaphylacticshock is favorably treated by the methods of the present invention.Further, it is contemplated that the methods of the present inventioncan be used to treat rheumatoid arthritis, lupus, and other inflammatoryand/or autoimmune diseases, heightened inflammatory states due toimmunodeficiency, e.g., due to infection with HIV, andhypersensitivities.

Wound Healing

Based on the anti-inflammatory properties of HO-1 and heme degradationproducts, the present invention contemplates that the methods describedherein can be used to promote wound healing (e.g., in transplanted,lacerated (e.g., due to surgery), or burned skin). They would typicallybe applied locally to the wound (e.g., as a wound dressing, lotion, orointment), but could be delivered systemically as well.

Reproductive Disorders

The present invention contemplates that the methods described herein canalso be used to treat or prevent certain reproductive disorders, e.g.,impotence and/or inflammation associated with sexually transmitteddiseases. Further, the methods of the present invention can be used toprevent premature uterine contractions, and can be used to preventpremature deliveries and menstrual cramps.

EXAMPLES

The invention is illustrated in part by the following examples, which isnot to be taken as limiting the invention in any way.

Example 1 Acute Colitis

In these experiments, CoPP, biliverdin, CO, and DFO were tested in anacute colitis animal model.

Materials and Methods

Animals. Pathogen-free male C57BL/6 mice, 4-6 weeks of age (Taconic;C57BL/6×129 svj strain) were used in this example. Mice were kept forone week at 4 mice/cage and fed normal laboratory chow and drinkingwater ad libitum prior to the experiments. All mice weighed between 22.5g and 27.7 g at the beginning of the trial. The animal experimentationprotocol was reviewed and approved by the Animal Care and Use Committeesof the Beth Israel Deaconess Medical Center.

Induction of Colitis. Colitis was induced by feeding mice 5% (wt/v)dextran sodium sulfate (DSS) (MW 40,000; ICN Biomedicals Inc., OH),dissolved in distilled water, for a period of 7 days. The resultingcondition was termed “DSS-colitis.” Control animals were fed distilledwater ad libitum. The mortality rate in the control group and theCO-treated group was 10% (2/20) and 14% (2/14), respectively. Theseanimals were excluded from the statistical analysis. In the othergroups, no mortality occurred during 7 days of 5% DSS treatment.

Experimental Reagents and Treatment Protocol. Cobalt protoporphyrin(CoPPIX), zinc protoporphyrin (ZnPPIX, Porphyrin Products, Logan, Utah)and biliverdin dihydrochloride (ICN Biomedicals Inc., OH) were dissolvedin a small amount 0.2 M NaOH, subsequently adjusted to a pH of 7.4 with1 M HCl, and diluted in 0.9% NaCl. The stock solutions (COPPIX andZnPPIX=1 mg/ml, biliverdin=10 mM) were aliquoted and kept at −70° C.until used. Light exposure was limited as much as possible. CoPPIX (5mg/kg), ZnPPIX (20 mg/kg) and biliverdin (50 μmol/kg) were adrninisteredto the mice intraperitoneally (i.p.) 24 hours before induction ofDSS-colitis and daily thereafter. Desferoxamine (DFO) was prepared inimidazole buffer (50 mM imidazole, 220 mM NaCl, pH 7.4). DFO wasinjected subcutaneously (s.c.) twice daily (125 mg/kg) or infused i.p.(130 mg/kg/day; pumping rate 0.5 μl/hour for 8 days) by miniosmoticAlzet™ pumps (Alza Corporation, Palo Alto, Calif.) (Postma et al., Exp.Parasitol. 89(3):323-30 1998). The pumps were installed i.p. 24 hoursbefore induction of colitis. When animals were sacrificed at day 7 ofDSS treatment, the pumps were removed and evaluated to determine if DFOapplication had taken place.

CO gas was administrated exogenously by putting animals in a CO exposurechamber (Otterbein et al., Nat. Med. 6(4):422-8, 2000). Briefly, CO at aconcentration of 1% (10,000 parts per million (ppm)) in compressed airwas mixed with balanced air (21% oxygen) in a stainless steel mixingcylinder before entering the exposure chamber. CO concentrations werecontrolled by varying the flow rates of CO in a mixing cylinder beforedelivery to the chamber. Because the flow rate is primarily determinedby the O₂ flow, only the CO flow was changed to deliver the finalconcentration to the exposure chamber. A CO analyzer (InterscanCorporation, Chatsworth, Calif.) was used to measure CO levelscontinuously in the chamber. Mice were placed in the chamber 24 hoursbefore the induction of the colitis and were kept in the exposurechamber during the whole period of the experiment (total of 8 days). COconcentration was maintained between 250 and 400 ppm at all times.Animals were removed daily from the chamber to assess weight and stool.

Evaluation of Symptoms of Colitis. All animals were evaluated clinicallyon a daily basis. The evaluation of each animal included a measurementof weight, hemoccult positivity and examination of stool for thepresence of gross blood and stool consistency. The disease activityindex (DAI) was calculated by scoring percent weight loss, intestinalbleeding (no blood; occult blood=hemoccult+; gross blood) and stoolconsistency (normal stool=well-formed pellets; loose stool=pasty andsemiformed stools; diarrhea=liquid stool that stuck to the anus) (Table1). TABLE 1 Scoring of the Disease Activity Index (DAI) Score Weightloss Stool consistency Bleeding 0 None Normal Normal 1  0-10% 2 10-15%Loose stools Hemoccult+ 3 15-20% 4   >20% Diarrhea Gross Bleeding

The animals were sacrificed, and the whole colon from the colo-cecaljunction to the anal verge was removed and gently cleaned of stool. Thedistal part of the colon was fixed in 10% formaldehyde and embedded inparaffin for staining with hematoxylin and eosin. The transversesections were graded in blinded fashion for severity of mucosal injuryon a scale of 0-4 as follows: Grade 0: intact crypt; Grade 1: loss ofthe basal one-third of the crypt; Grade 2: loss of the basal two-thirdsof the crypt; Grade 3: loss of the entire crypt with the surfaceepithelium remaining intact; Grade 4: loss of both crypt and surfaceepithelium (erosion). In addition, the percentages of the respectiveinjured surface areas for each tissue section were scored on a scale of1-4 as follows: 1=1% to 25%; 2=26% to 50%; 3=51% to 75%; and 4=76% to100%. The product of the two scores gave the crypt score for eachsection. The means of all sections were then calculated for each animal.

Western Blot Analysis. Tissue samples from the distal colon of theanimals were removed and snap frozen with liquid nitrogen. The frozentissue was ground thoroughly and homogenized in Ripa buffer supplementedwith proteinase inhibitors. The protein concentration was determined bythe Bio-Rad Dc Protein Assay™ according manufacturers instructions(Bio-Rad, Hercules, Calif.). Electrophoresis was performed underdenaturing conditions according to Laemmli with 10% polyacrylamide gelsby loading 35 μg protein. Proteins were transferred onto apolyvinyldifluoridine membrane (Immobilon P™; Millipore, Bedford, Mass.)by electroblotting. Proteins were then detected with rabbit polyclonalantibodies directed against human HO-1 (StressGen, Victoria, Canada) orβ-tubulin (Boehringer Mannheim, Mannheim, Germany). Proteins werevisualized using HRP-conjugated donkey anti-rabbit IgG or goatanti-mouse IgG (Pierce) and the ECL assay (Amersham Life Science,Arlington Heights, Ill.), according to manufacturer's instructions.

Semi-Quantitative PCR. RNA was extracted using RNeasy Mini Kits (QiagenInc., CA, USA) and reverse transcribed into cDNA with the RNA PCR Kit(TaKaRa, PanVera, Madison, Wis., USA). A total of 2 μl of cDNA wasamplified in a 50 μli reaction mix containing 10 μM dNTPs, 50 pg of5′-prime and 3′-prime oligos, 2.5 U of LA-Taq polymerase (TaKaRa) andMgC12, specific to each primer pair used. The primers for murine andhuman HO1 (372 bp) (5′: TGA AGG AGG CCA CCA AGG AGG T (SEQ ID NO:1); 3′:AGG TCA CCC AGG TAG CGG GT (SEQ ID NO:2)) and β-actin (525 bp) (5′: GCCATC CTG CGT CTG GAC CTG G(SEQ ID NO:3); 3′: TAC TCC TGC TTG CTG ATC CACA (SEQ ID NO:4)) were obtained from Life Technologies, NY, USA. PCRreactions were performed after a 4 min denaturation at 94° C. arepeating the cycle 94° C., 55° C. and 72° C. each for 1 min for numberof cycles specific for each primer pair in a Peltier Thermal CyclerPTC-200 (MJ Research, Las Vegas, Nev., USA). PCR products (10-20 μl)were analyzed in an ethidium bromide-stained 1% agarose gel.

Statistical Analysis. Percent body weight loss and DAI score data weresummarized as mean±standard deviation of mice untreated or treated withCoPP, ZnPP, biliverdin, CO or DFO. Significance was calculated using theMann-Whitney test and defined as p<0.05. Time to occurrence of symptomswas calculated using Kaplan-Meier life tables. Differences betweengroups were tested using a log-rank test and the mean time tooccurrence, with a 95% confidence interval reported.

Results

DSS Induces Acute Hemorrhagic Colitis and HO-1 Expression. The C57/BL6mice exposed to 5% DSS in the drinking water showed the first clinicalsymptoms by days 2 and 3, with the development of loose stools andhemoccult positive stools. By days 5 to 6, most of the animals developedthe complete picture of a hemorrhagic colitis with diarrhea and grossbleeding. In the control group, 2 deaths occurred (out of 20 subjects,10%) before the termination of the 7-day trial. Both of thesemortalities occurred on day 6. These animals were excluded from thestatistical analysis.

Induction of DSS-colitis leads to a marked elevated level of HO-1 mRNAover time (FIGS. 1A-1B). An elevated level of HO-1 protein expressionwas also observed (FIG. 1C). After only 24 hours, a slight increase inHO-1 protein level was visible; it thereafter increased consistently tomaximum levels at 7 days.

Treatment with CoPP Induces HO-1 in the Intestinal Tissue. Dailyadministration of CoPP (5 mg/kg) i.p. (starting 24 hours before exposingthe animals to DSS via the drinking water) induced HO-1 in the colonictissue of the animals. The CoPP-treated animals showed consistently highlevels of HO-1 protein in the intestine over the entire 7 day period ofthe experiment, whereas non-treated animals exhibited slowly increasingHO-1 levels that reached a maximum at day 7 (FIGS. 2A and 2B versusFIGS. 1A and 1B). The level of HO-1 in the non-treated animals at day 7was below the level of HO-1 observed in the CoPP-treated animals.

HO-1 Induction Ameliorates DSS-Colitis and Reduces Associated ColonicLesions. The constant induction of HO-1 by CoPP treatment amelioratedDSS-colitis as observed clinically and morphologically. At day 7, themean total percent body weight loss was significantly lower in theCoPP-treated group (n=12) as compared to the non-treatment group (n=20)(−11.8% versus −22%; p<0.001; FIG. 3A). Further, the DAI, which factorsin weight loss, stool consistency, and intestinal bleeding, showed asignificant difference between the two groups (2.8 with CoPP-treatmentand 3.8 in non-treated animals, p<0.001; see FIG. 3B). During the courseof disease development, a significant difference in the DAI betweenCoPP-treated and untreated groups was observed begning on day 2(difference in DAI=0.4, p<0.05). Maximum protection conferred by CoPPoccurred at day 5 (difference in DAI=1.64; p<0.001). Toward the end ofthe 7 days, the difference had lessened (difference in DAI=1.04;p<0.001; FIG. 3C). The time required for animals to develop symptoms,e.g., occurrence of loose stool and gross intestinal bleeding, wasprolonged significantly in the group in which HO-1 was induced with CoPP(p<0.02 and p<0.005, respectively; FIGS. 3D and 3E). Animals treatedwith CoPP developed loose stool at a mean of 4.6 days (95% confidenceinterval: 4.1 to 5.1 days) as compared to non-treated animals, whichdeveloped loose stool at 3 days (95% confidence interval 2.4 to 3.6days). Bleeding was observed in CoPP-treated animals after a mean of 6.9days (95% confidence interval: 6.3 to 7.4 days) as compared 5.1 days fornon-treated animals (95% confidence interval: 4.6 to 5.6 days). Acontrol group treated with zinc protoporphyrin (ZnPP), a blocker of HO-1enzymatic activity, showed no significant changes in the natural courseof DSS colitis as compared to untreated animals (FIGS. 2A-2B). Thus, itappears that the protective effect of CoPP is based on the specificinduction of HO-1 by this compound.

Histologically, the animals with DSS-colitis alone showed progressiveloss of the cryptal structure, which led to complete destruction of themucosal glands by day 7 (FIG. 4A). Mixed inflammatory infiltrationconsisting of mainly macrophages and neutrophils and some lymphocytesappeared in the lamina propria and submucosa. Sporadically, cryptabscess and erosions of the surface epithelium were observed (FIG. 4A).In contrast, CoPP treatment samples showed remaining cryptal structuresand intact epithelial surface, although increasing separation betweenthe base of the crypt and muscularis mucosa, along with mildinflammatory infiltrate, was observed at day 7 as well (FIG. 4A).Evaluation of the colonic damage revealed that the CoPP group had asignificantly lower crypt score in comparison to the non-treatment group(2.6 versus 4.8; p<0.05), which indicates less extensive and severedestruction of the mucosal glands (FIG. 4B). The clinical diseaseactivity supported by the pathologic changes showed a significantlybetter course and outcome of the CoPP treated animals.

Exogenously Applied Biliverdin Ameliorates DSS-Colitis. The protectiveeffects of biliverdinibilirubin, iron/ferritin, and CO againstDSS-colitis were investigated. In the first group (n=12), dailytreatment with biliverdin injected i.p. was found to be protectiveagainst DSS-colitis. Percent weight loss (FIG. 5A) and the DAI (FIG. 5B)were significantly reduced after 7 days as compared to control animals(percent weight loss: 16.5% versus 22%; DAI score 3.3 versus 3.8;p<0.01). As with CoPP treatments, the time to the development ofsymptoms (e.g., loose stool and gross intestinal bleeding) was prolongedsignificantly (p<0.01 and p<0.05, respectively) (FIGS. 5D and 5E).Animals treated with biliverdin developed loose stool at a mean of 4.4days (95% confidence interval: 4 to 4.9 days) as compared to non-treatedanimals at 3 days (95% confidence interval: 2.4 to 3.6 days). Bleedingoccurred in biliverdin-treated animals after a mean of 6.2 days (95%confidence interval: 5.4 to 6.9 days) as compared to non-treatedanimals, which exhibited bleeding at 5.1 days (95% confidence interval:4.6 to 5.6 days).

Using light microscopy, it was observed that the biliverdin treatmentgroup had a preserved cryptal structure (FIG. 6A). The crypt score wasalso significantly better in comparison to the non-treatment group (3versus 4.8; p<0.05; FIG. 6B). Overall, biliverdin did not mediateprotection to the same extent as CoPP/HO-1, but the difference betweenthe effects of CoPP/HO-1 and biliverdin did not reach significance.

The continuous exposure of animals to 200-400 ppm CO (200 ppm, n=6; 400ppm, n=6) did not have an effect compared to the control (FIGS. 5A-B).

DFO, an iron chelator, was used to assess the potential function ofendogenous ferritin. Subcutaneous injections of 125 mg/kg DFO (n=4)administered twice daily to animals showed no protective effect.Further, intraperitoneally-placed osmotic pumps (n=4) were also used.The pumps delivered 130 mg/kg/d DFO at a constant pumping rate of 0.5μl/hour. While this dose has previously been proven to efficientlyremove endogenous iron from mice (Postma et al., Exp. Parasitol.89(3):323-30, 1998), no protective effect was observed (FIGS. 5A-5B).

Conclusions

These results indicate that upregulation of HO-1, e.g., byadministration of CoPP or biliverdin, alone or in combination with CO,is useful in the treatment of colonic inflammation, e.g., colitis andassociated colonic lesions.

Example 2 Cardiac Transplantation

In these experiments, CoPP, biliverdin, and CO were tested in a cardiactransplantation mouse model.

Materials and Methods

Animals. Male DBA/2 (H-2^(d)), B6AF1 (H-2^(k/d,b)) and FVB (H-2^(q))mice were purchased from Jackson Lab. Inc. (Bar Harbor, Me.). They weremaintained in the institutional specific-pathogen free facility, whichhas an appropriate light cycle with free access to water and chow adlibitum, and used for experiments at age of 6-10 weeks.

Cardiac transplantation. DBA2/J and B6AF1 mice were used as donor andrecipient, respectively. Heterotopic heart transplantation was carriedout according to the procedure of Corry and Russell (Corry et al.,Transplant Proc. 5(1):733-5, 1973). In brief, the heart was excised fromdonor mice after ligation of the pulmonary vein, and the inferior andsuperior vena cavas. Under a microscope, the graft aorta and pulmonaryartery were anastomosed to the recipient's abdominal aorta and inferiorvena cava, respectively. Secondary heart grafting into the neck wasperformed using a cuff technique as described previously (Matsuura etal., Transplantation 51(4):896-8, 1991). Briefly, the heart graft washarvested from either DBA2/J (donor strain) or FVB (third-party strain)mice. The right jugular vein and the right common carotid artery ofrecipient were dissected, and a cuff (polyethylene or polyimide) wasconnected to these vessels. The graft aorta and pulmonary artery weresleeved over the cuff on the recipient's common carotid artery and thejugular vein, respectively, and fixed with a ligature. Aftertransplantation, beating of the graft was monitored by daily palpation,and scored +1 to +4 according to the strength of graft contractions.Graft rejection was defined as cessation of beating, and was confirmedby direct inspection followed by histological examination.

Cells and Culture Medium. Primary murine leukocytes were isolated bymincing the spleen followed by osmotic lysis of red blood cells. T cellswere further enriched by passing through a nylon-wool mesh column, andpurified using the MACS Pan T cell isolation kit (Miltenyi Biotec Inc.,Auburn, Calif.) according to the manufacture's instructions. Purity ofCD3+ T cells was more than 95%, as determined by flow cytometry. TheJurkat cell line was maintained in RPMI 1640 culture media (BioWhittaker Inc., Wallcersville, Md.) supplemented with 2 mM 1-glutamine,100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum.For murine primary cell culture, 2-ME (50 μM) was also added to themedia.

Experimental Reagents and Treatment ProtocoL Cobalt protoporphyrin(CoPPIX), zinc protoporphyrin (ZnPPIX, Porphyrin Products, Logan, Utah)and biliverdin dihydrochloride (ICN Biomedicals Inc., OH) were dissolvedin a small amount 0.2 M NaOH, subsequently adjusted to a pH of 7.4 with1 M HCl, and diluted in 0.9% NaCl. The stock solutions (CoPPIX andZnPPIX=1 mg/ml, biliverdin=10 mM) were aliquoted and kept at −70° C.until used. Light exposure was limited as much as possible.

Donor and recipient mice were treated either with CoPPIX (5 mg/kg/day),ZnPPIX (5 mg/kg/day) or biliverdin (administered once, twice or threetimes per day at a dose of 50 μmol/kg/dose). All reagents wereadministered by intraperitoneal (i.p.) injection. Donor animals weretreated for 2 days, starting from 2 days before graft harvest (day-2 andday-1). Treatment of recipient mice was initiated one day beforetransplantation, and continued until 13 days post-transplant (day-1 today 13). For the experiments using donor splenocyte infusion (DSI) onday-7 (DSI (D-7)), treatments of recipient animals (either with CoPPIXor ZnPPIX) started from day-8 and were terminated on day 6. Recipientsreceived no further treatment. Spleen cells (2×10⁷) isolated from DBA/2Jmice as described herein were used for DSI treatment. The cells wereinjected via the penile vein with 200 μl of 0.9% saline solution.

Bilirubin assay. Biliverdin at 50 μmol/kg was injected i.p. into B6AF1mice. Blood was drawn before and 15, 30, 60, 120, 240, and 360 minutesafter biliverdin injection. Serum was collected by centrifugation ofblood samples, and the total bilirubin level was measured using a totalbilirubin assay kit (Sigma Aldrich, St. Louis, Mo.). Measurement oftotal bilirubin was performed according to the kit protocol, and wasduplicated for each sample. This experiment was repeated four times.

Con A and anti-CD3 mAb mediated proliferation assay. B6AF1 splenocytesor purified T cells were prepared according to the herein-describedmethod and used as responder cells. Responders (2.5×10⁵/well) werestimulated with either Con A (1 μg/ml) or anti-CD3 mAb (1 μg/ml) andcultured in a 96 well round bottom plates. Purified T cells (5×10⁴/well)were cultured in anti-CD3 mAb (10 μg/ml) coated 96 well flat bottomplates in the presence of anti-CD28 mAb (1 μg/ml). Cells were culturedwith or without biliverdin (at various concentrations) for 48 hours at37° C., 95% air with 5% CO₂. They were pulsed with ³H-thymidine (1μCi/well) 16 hours before termination of cell culture and ³H-thymidineincorporation was measured by using a β-counter. These proliferationassays were performed in triplicate and were repeated 3 times.

Mixed lymphocyte culture (MLC). Irradiated (25Gy, ¹³⁷Cs) DBA/2splenocytes (5×10⁵/well) were co-cultured with responders (5×10⁵/well)in a 96 well round bottom plates for 3 days at 37° C., 95% air with 5%CO₂. When using cells from cardiac recipients as responders, theco-culture period was 3 days without any further in vitro treatment.³H-thymidine (1 μCi/well) was added 16 hours before termination of cellculture, and ³H-thymidine incorporation was measured.

IL-2 assay. B6AF1 leukocytes (2.5×10⁵ cells/well) were stimulated byanti-CD3 mAb (1 μg/ml), and cultured in 96 well round bottom plates withor without biliverdin (50 or 100 μM). Forty-eight hours later, culturesupernatant (100 μl) was collected and stored at −80° C. until use. Allcultures were performed in triplicates. IL-2 levels in the supernatantswere measured by enzyme-linked immunosorbent assays (ELISA) using anIL-2 assay kit (Quantikine® M, R&D Systems, Inc), following kitinstructions. Measurements of each supernatant sample for IL-2 wereperformed in duplicate. The experiment was repeated 4 times.

Flow cytometric analysis (IL-2R expression). B6AF1 leukocytes (5×10⁵cells/well) were stimulated with anti-CD3 mAb (1 μg/ml) and cultured in96 well round bottom plates with or without biliverdin (100 μM). Six and24 hours after stimulation, cells were harvested, washed, and stainedwith fluorochrome conjugated isotype control Abs, or specific anti-CD4and/or anti-CD25 mAbs (all antibodies obtained from BD Pharmingen).Non-stimulated naïve leukocytes were used as a negative control.Following Abs staining, cell samples were analyzed using a FACSort™ flowcytometer and CellQuest™ software (BD Biosciences, Palo Alto, Calif.).Ten thousand CD4+ T cells were acquired for each sample and their CD25expression was examined. The experiment was repeated 3 times.

HO activity assay. DBA/2J mice were given either no treatment or CoPPIXor ZNPPIX at a dose of 5 mg/kg, i.p. (n=10 per group). One day after thetreatment, the animals were sacrificed, the spleen and the heart wereexcised, and tissue samples were frozen at −80° C. Frozen tissue sampleswere homogenized in ice-cold sucrose and Tris-HCl buffer. The microsomalpellet was obtained after centrifugation and re-suspended inMgCl₂-potassium phosphate buffer. Sample protein was then incubated withthe reaction mixture containing rat liver cytosol, hemin,glucose-6-phosphate, glucose-6-phosphate dehydrogenase and NADPH(Sigma-Aldrich Corporation, St. Louis, Mo.) for 60 minutes at 37° C. Thegenerated bilirubin was measured by reacting with diazotized surfanilicacid to yield azobilirubin using spectrophotometer.

Protein extraction and Western blot. DBA/2 mice were injected i.p. withCoPPIX at a dose of 5 mg/kg. Non-treated animals served as naïvecontrols. One, 2, 4, and 7 days following treatment, the heart and thespleen were excised from the animals, and samples were snap-frozen inliquid nitrogen. Protein extracts were prepared from the obtainedtissues, electrophoresed under denaturing conditions (10% polyacrylamidegels) and transferred onto polyvinyldifluoridine membranes (Immobilon™P, Milpore, Bedford, Mass.). HO-1 was detected using the rabbitanti-human HO-1 polyclonal antibody (Stress Gen Biotechnologies Corp.,Victoria, Canada); mactin was detected using the goat anti-mouse mactinmAb (1A4, Sigma-Aldrich Corporation). Primary antibodies were detectedusing horseradish peroxidase conjugated donkey anti-rabbit or goatanti-mouse IgG secondary antibodies (Pierce, Rockford, Ill.). Proteinwas visualized using the Enhanced Chemi Luminescence(ECL™) Assay kit(Amersham Life Science Inc., Arlington Heights, Ill.), according to themanufacturer's instructions, and stored in the form of photo radiographs(Biomax™ MS, Eastman Kodak, Rochester, N.Y.). The amount of HO-1expression was normalized with mactin expression, and was quantified byusing the ImageQuant™ software (Molecular Dynamics, Sunny Vale, Calif.).

Nuclear protein extraction and electrophoretic mobility shift assay(EMSA). Jurkat T cells were cultured in 10% FCS supplemented RPMI 1640with 50 nM phorbol myristyl acetate (PMA) and 2 μM ionomycin (bothreagents from ICN Biomedicals) in the presence or absence of 100 μMbiliverdin. Following 0, 2, and 4 hrs of stimulation, cells (30×10⁶cells for each group at each time points) were collected, washed twicewith PBS, and pelleted. Nuclear and cytoplasmic extracts were preparedaccording to a modified Shapiro's method (Shapiro et al., DNA7(1):47-55, 1988). Protein concentrations were determined by theBradford assay. The following oligonucleotides (hvitrogen, Calsbad,Calif.) were used for EMSA: NFAT, (coding)5′-GCCCACAGAGGAAAATTTGTTTCATACAG-3′ (SEQ ID NO:5), (non-coding)5′-CTGTATGAAACAAATTTTCCTCTGTCCGC-3′ (SEQ ID NO:6); and NF-κB, (coding)5′-AGCTTAGAGGGGACTTTCCGAGAGGA-3′ (SEQ ID NO:7), (non-coding)5′-TCCTCTCGGAAAGTC-CCCTCTAAGCT-3′ (SEQ ID NO:8). Qligonucleotides wereradioactively labeled with [³²P] ATP. The EMSA reactions were assembledas previously described (Usheva et al., Proc. Natl. Acad. Sci. USA93(24):13571-6, 1996). Digital images from the EMSA radiographs wereobtained using an image scanner.

Statistics. Cardiac graft survival was plotted by the Kaplan-Meiermethod, and a log-rank test was applied to compare statisticalsignificance. Expression levels of HO-1/α-actin protein are expressed asmean±standard deviation. Other data are expressed as mean±standard errorof the mean (mean±SEM). Intergroup statistical analysis was performed byone-way ANOVA, and Fisher's PSLD was used for a post-hoc test. Acomparison was considered statistically significant when the p-value was<0.05.

Results

Induction of HO-1 expression and enzymatic function prolongs cardiacallograft survival. As is shown in FIGS. 7A-7C, administration of CoPPIXup-regulated HO-1 protein expression in the heart and spleen of adultmice. Maximal HO-1 expression was detected one day after CoPPIXinjection (FIG. 7A). HO-1 enzymatic activity was also significantlyenhanced by CoPPIX, while this was not the case for ZNPPIX, which isknown to inhibit HO-1 function (FIG. 1B). Based on these data, theeffect of induction of enzymatically active HO-1 expression by CoPPIX oncardiac allograft rejection was evaluated. ZNPPIX was used as a controlreagent. Both CoPPIX and ZNPPIX were administered daily to the donorfrom day-2 and to the recipient from day-1 to day 13 post-transplant.Untreated B6AF1 recipients rejected DBA/2 cardiac allografts at a mediansurvival time (MST) of 11.5 days (FIG. 7D). Induction of HO-1 expressionby CoPPIX administration resulted in a significant prolongation of graftsurvival (p<0.005 versus control). Two of 6 grafts (33.3%) survivedlong-term, i.e., >100 days, while the MST of the other 4 rejected graftswas 18 days. In contrast, administration of ZnPPIX did not result inprolongation of graft survival as compared to untreated controls; thesegrafts were rejected promptly with a MST of 11 days.

Biliverdin induces donor specific tolerance to cardiac allograft. As isshown in FIGS. 8A-8C, administration of biliverdin (50 μmol/kg) usingthe same treatment schedule as CoPPIX, i.e., one dose per day, prolongedgraft MST to 20.5 days (p<0.01 when compared to control) (FIG. 8A). Todetermine the approximate half-life of exogenously administeredbiliverdin, serum bilirubin level was analyzed after a single 50 μmol/kginjection. Upon administration of biliverdin, serum bilirubin levelspeaked rapidly to reach a maximal level at 15 minutes with a return tobasal levels 4 to 6 hours thereafter (FIG. 8B). We thus tested theeffects of administering biliverdin two (every 12 hours) or three (every8 hours) times per day at 50 μmol/kg per dose. These two treatmentschedules significantly increased graft survival, with 4 of 6 (66.7%)grafts surviving for more than 100 days (both p<0.001 versus control,FIG. 2A). These recipients who accepted the allografts for long-term bybiliverdin treatment were challenged by second set transplantation usingcardiac allografts from either the donor (DBA2/J) or third party (FVB)strain mouse (n=3 for each strain). As shown in the FIG. 8C, therecipients harboring the initial graft accepted the secondary heartgraft from the donor strain for more than 60 days, whereas they rejectedthird party graft within 11 days. These data suggest that administrationof biliverdin is capable of inducing tolerance in the MHC class I plusclass II mismatched mouse heart transplantation.

Induction of HO-1 expression or biliverdin administration suppresses theT cell mediated alloimmune response in vivo. As shown in FIG. 9, theimmune response of cardiac allograft recipients under differenttreatments, i.e., no treatment (n=4), CoPPIX (n=4), ZNPPIX (n=4) orbiliverdin (50 μmol/kg/lx daily and 50 μmol/kg/3× daily; n=4 for eachgroup) was assessed. Recipient splenocytes harvested 5 days aftercardiac transplantation were used as responder cells in an in vitroculture with DBA/2 splenocytes as feeder cells. Proliferation wasmeasured at 72 hours (FIG. 9). HO-1 induction by CoPPIX in vivoinhibited splenocyte proliferation in a significant manner, as comparedto controls (p<0.05), whereas ZnPPIX administration did not. Theproliferation of the splenocytes from biliverdin-treated recipients wassignificantly suppressed, both in recipients receiving one and in thosereceiving three doses of biliverdin per day, when compared to thecontrol (p<0.01).

Biliverdin suppresses T cell proliferation in vitro. To assess whetherbiliverdin has a suppressive effect on T cell proliferation in vitro,naïve B6AF1 splenocytes were stimulated with ConA, anti-CD3 mAb orirradiated DBA/2 splenocytes in the presence or absence of biliverdin.Biliverdin suppressed leukocyte proliferation in all cases in adose-dependent manner (FIGS. 10A-10C). A significant suppressive effectwas achieved at concentrations of 50 μM and 100 μM for ConA or anti-CD3mAb driven T cell proliferation and at 10 μM in the case of alloantigenmediated T cell activation (FIG. 10C). Further, the suppressive effectof biliverdin appeared to act directly on T cells in that proliferationof purified B6AF1 mouse T cells (purity over 95%) in response toanti-CD3 mAb plus anti-CD28 mAb co-stimulation, a combination shown tostimulate T cells in an antigen presenting cell independent manner, wasalso suppressed by biliverdin (FIG. 10D).

Biliverdin suppresses IL-2 production by blockade of nucleartranslocation of NFAT and NF-κB in T cells. IL-2 production bysplenocytes following anti-CD3 mAb stimulation was significantlysuppressed in a dose-dependent manner by biliverdin treatment in vitro(FIG. 11A). Addition of exogenous recombinant IL-2 (50 U/ml) intoculture wells 6 or 24 hours after biliverdin treatment overcame thesuppressive effect of biliverdin (FIG. 11B). Moreover, biliverdin at aconcentration of 100 μM did not affect the expression of the IL-2receptor α-chain (IL-2Rα, CD25) of anti-CD3 mAb stimulated splenic CD4+T cells. Representative results are shown after 6 hours of culturecompared to the non-treatment control (FIG. 11C); similar results wereseen after 24 hours. As one theory, not meant to be limiting, biliverdinmay exert its effect on T cells by interfering directly with the signaltransduction pathway leading to IL-2 synthesis, but not bydown-regulation of IL-2Rα surface expression or blocking the signalingpathways involved in IL-2 driven proliferation.

To investigate whether the activation of transcription factors involvedin IL-2 transcription/expression, i.e., NF-κB and NFAT, were modulatedby biliverdin, Jurkat T cells were stimulated by the combination of PMAand ionomycin. The level of nuclear translocation of NF-κB and NFAT wasexamined by measuring DNA-binding activity of nuclear extracts by EMSA.PMA plus ionomycin stimulation induced the activation of NF-κB and NFATnuclear translocation and DNA binding, as assessed 2 and 4 hours afterstimulation, respectively. Biliverdin inhibited DNA binding of bothNF-κB and NFAT (FIG. 11D), suggesting that it suppresses IL-2 productionvia inhibition of its transcription, possibly by blocking NFAT and NF-κBactivation.

Induction of HO-1 by CoPP cooperates with donor splenocyte infusion(DSI) to achieve long-term acceptance of cardiac allografts. When DSIwas given to a B6AF1 recipient immediately after transplantation (day0), a DBA/2J heart allograft was rejected promptly, similar to theuntreated control (FIG. 12A). Induction of HO-1 expression by CoPPIXadministration to the donor from day-2 and to the recipient from day-1to day 13 post-transplant together with DSI (D0) significantly (p<0.001)prolonged graft survival (7 of 8 (87.5%) survived long-term for morethan 100 days) when compared to DSI (D0) or CoPPIX treatment alone.Conversely, treatment with ZnPPIX plus DSI (D0) had no significant graftprolongation effect as compared to the control or DSI (D0) alone. Thesedata suggest that induction of HO-1 expression helps to achievelong-term allograft survival.

Enzymatic activity of HO-1 is essential for donor splenocyte infusion(DSI) mediated long-term allograft acceptance. DSI alone given 7 daysbefore heart grafting (D-7) led 50% (4/8) of the allografts to survivefor more than 100 days (FIG. 12B). The induction of HO-1 expression byadministration of CoPPIX to the donor for 2 days (day-1 and day-2) andto the recipient from day-8 (one day before DSI) to day 6 plus DSI (D-7)allowed all of the cardiac allografts (100%, 7/7) to survive over 100days. The treatment period and the dose are identical to other CoPPIXtreatment protocols used in the present study. Importantly, when ZnPPIX,an inhibitor of HO-1 enzymatic activity, was administered to both donorand recipient under the same protocol used for the CoPPIX treatment asmentioned former, the graft prolongation effect of DSI (D-7) wascompletely abrogated such that none (0/9) of allografts underwentlong-term survival (MST: 13 days). These results suggest the importanceof HO-1 activity upon DSI mediated long-term allograft survival.

Conclusions

These results indicate that the administration of CoPPIX and/orbiliverdin can contribute to long-term allograft survival. The effect ofCoPPIX administration appears to synergize with the administration ofDSI, suggesting that treatment to induce HO-1 expression, e.g., byadministration of CoPPIX, in addition to DSL would further increaseallograft survival.

Example 3 Over-Expression of H-Ferritin Protects Rat Livers fromIschemia Reperfusion Injury (IRI) and Prevents Hepatocellular Damageupon Transplantation into Syngeneic Recipients

Expression of the ferritin heavy chain (H-ferritin) gene was evaluatedfor potential cytoprotective effects that could be used in a therapeuticmanner, e.g., to suppress ischemia reperfusion injury (IRI) oftransplanted organs.

Materials and Methods

TNF-α induced apoptosis. Prinary bovine aortic endothelial cells (BAEC)or murine 2F-2B EC (ATCC) were cotransfected with β-galactosidase pluscontrol (pcDNA3 or pcDNA3/HO-1) or pcDNA3/H-Ferritin. Apoptosis wasinduced by TNF-α plus Act.D and quantified.

Etoposide and serum deprivation-induced apoptosis. Murine 2F-2B cellswere cotransfected with β-galactosidase plus control (pcDNA3) orpcDNA3/H-ferritin and treated with etoposide (200 μM, 8 h) or subjectedto serum deprivation (0.1% FCS, 24 hours).

H-ferritin toxicity. Murine 2F-2B cells were cotransfected withβ-galactosidase plus increasing amounts of pcDNA/H-ferritin (0.1-200ng/well). As controls, pcDNA3 and pcDNA3/HO-1 were used. EC apoptosiswas induced by TNF-α plus Act.D, and the apoptosis ofβ-galactosidase-transfected EC was quantified.

Cold ischemia in vitro model Livers were harvested from SD rats, exposedto ischemia (24 hours, 4° C., University of Wisconsin (UW) solution(Oxford Textbook of Surgery, Morris and Malt, Eds., Oxford UniversityPress, 1994) and perfused ex vivo with syngeneic blood. Livers weretransduced with recombinant adenovirus encoding H-ferritin, and controlswere either non-transduced or transduced with β-galactosidase. Bileproduction was measured using standard methods at 30, 60, 90 and 120minutes.

Cold ischemia in vivo orthotopic transplantation model Livers wereharvested from SD rats, exposed to ischemia (24 hours, 4° C., Universityof Wisconsin (UW) solution (Oxford Textbook of Surgery, Morris and Malt,Eds., Oxford University Press, 1994) and perfused ex vivo with syngeneicblood. The livers were then transduced with recombinant adenovirusencoding H-ferritin (controls were either non-transduced or transducedwith β-galactosidase) and transplanted into syngeneic recipients. Eightto ten animals were analyzed per group.

Results

H-ferritin protects endothelial cells from undergoing apoptosis. ControlEC (transfected with pcDNA3) showed 60-70% apoptotic cells afterexposure to TNF-α in the presence of the transcription inhibitorActinomycin D (Act.D) (FIG. 13A-13C). Expression of H-ferritinsuppressed TNF-α mediated EC apoptosis (10% apoptotic EC) (FIG.13A-13C). This protective effect was observed in the EC line 2F-2B aswell in primary bovine aortic EC (BAEC) (FIG. 13A). The anti-apoptoticeffect of H-ferritin was also observed with other pro-apoptotic stimulisuch as etoposide or serum deprivation (FIG. 13B). This effect wasdose-dependent, showing protection from 1 ng to 100 ng of thepcDNA3/H-ferTitin expression vector per 3×10⁵ cells (FIG. 13C). Levelsof pcDNA3/H-ferritin over 100 ng may be toxic as evidenced by anincreasing number of apoptotic cells in TNF-α/Act.D stimulated cells aswell as in control cells treated with pcDNA3/H-ferritin plus Act.D only(FIG. 13C).

Recombinant adenovirus mediated H-ferritin expression protects liversfrom ex-vivo reperfusion iniurv. Rat livers exposed to prolonged coldischemia (UW solution, 4° C., 24 hours) showed severe signs of injuryonce re-perfused ex-vivo with whole syngeneic blood. Injury wasevidenced by the relative low increase in portal blood flow (from0.63±0.076 ml/min/g at time 0 to 1.13±0.23 at ml/min/g at 120 min) andbile production (from 0.00338±0.0078 ml/g at time 0 to 0.025±0.01 mug at120 min)(FIG. 14A) following reperfusion as well as by a significantincrease in ALT release (from 7.2±4.9 IU/l at time 0 to 173±71 IU/l at120 min). Similar results were obtained in β-galactosidase recombinantadenovirus transduced livers in that there was a similar level of portalblood flow (from 0.626±0.079 m/min/g at time 0 to 1.035±0.105 ml/min/gat 120 min) and relative low level of bile production (from0.0005±0.0002 ml/g at time 0 to 0.03±0.0085 ml/g at 120 min) as well assignificant ALT release (from 12.5±10.5 IU/l at time 0 to 148±92 IU/l at120 min). Unlike untreated or β-galactosidase transduced groups,H-ferritin transduced livers showed significantly (p<0.01) greaterincreases in portal blood flow (from 0.62±0.099 ml/min/g at time 0 to1.3721±0.133 ml/min/g at 120 min) and bile production (from0.00621±0.0029 mug at time 0 to 0.043±0.0088 at 120 min)(FIG. 14A).Further, ALT release in the H-ferritin transduced livers remained atrelatively low levels (from 9.3±4.51 U/1 at time 0 to 68.6±14.8 IU/l at120 minutes). At 2 hours of reperfusion, myeloperoxidase activity, amarker of neutrophil mediated oxidative stress injury, was significantlyinhibited (p<0.05) in H-ferritin transduced livers (0.736±0.58 units/g),as compared to untreated (1.35±0.227 units/g) or β-galactosidasetransduced (3.12±0.9 units/g) livers. These results support the notionthat over-expression of H-ferritin protects livers from IRI despiteprolonged periods of cold ischemia. Livers transduced with theH-ferritin gene also had a significantly better preservation of theirhistological detail, as compared to non-transduced (p<0.05) orβ-galactosidase (p<0.05) transduced livers, as assessed by standardSuzuki's pathological scoring.

Recombinant adenovirus mediated H-ferritin expression prevents IRIfollowing orthotonic liver transplantation. Livers from SD rats exposedto prolonged cold ischemia (U.W. solution, 4° C., 24 hours) showedsevere signs of hepatocellular damage following transplantation intosyngeneic SD recipients. This was evidenced by the high serum levels ofAST (3928±1455 IU/L) 24 hours post-transplant. Similar results wereobtained in liver transplant recipients transduced with aβ-galactosidase recombinant adenovirus (4887±500 IU/L). In markedcontrast, in animals bearing liver transplants transduced with theH-ferritin recombinant adenovirus, AST release (1368±550.8 IU/L) wassignificantly inhibited as compared to β-galactosidase/untreated groups(p<0.05). That H-ferritin transduced livers were protected from IRI isstrongly supported by the demonstration that up to 90% of H-ferritintransduced livers survived for longer than 14 days when transplantedinto syngeneic SD recipients. In marked contrast, only 40-50% ofnon-transduced or α-galactosidase transduced livers survived longer than14 days when transplanted into syngeneic SD recipients (FIG. 14B). Therelative number of cells undergoing apoptosis in H-ferritin transducedlivers transplanted into syngeneic recipients was significantly reducedas compared to non-transduced or β-galactosidase-transduced liverstransplanted under the same conditions (FIG. 14B). Prolonged survival inrecipients receiving H-ferritin recombinant adenovirus-transduced liverswas significantly enhanced as compared to recipients transplanted withnon transduced or β-galactosidase recombinant adenovirus transducedlivers (p<0.001). This finding is consistent with the hypothesis thatthe anti-apoptotic effect of H-ferritin may contribute to its overallcytoprotective function in transplanted livers.

Model for the cytoprotective action of ferritin. As shown in FIG. 15,upon ischemia and reperfusion, free heme is internalized by EC. HO-1action on heme releases Fe²⁺, which catalyzes the conversion of hydrogenperoxide (H₂O₂) into OH⁻ and OH⁻, through the Fenton reaction. Thesetrigger signal transduction pathways that promote inflammation andapoptosis. Ferritin binds Fe²⁺ and prevents it from reacting with H₂O₂,thus blocling this process.

Conclusions

These results demonstrate an anti-apoptotic function of H-ferritin andsuggest that an increase in H-ferritin activity, e.g., by administrationof exogenous ferritin or by increased expression of the H-ferritin gene,can be used in a therapeutic manner. By its ability to suppress liverIRI, expression of the H-ferritin gene may result in the safer use ofliver transplants despite prolonged periods of cold ischemia. As onetheory, not meant to be limiting, the protective effect of H-ferritinappears to be associated with its ability to inhibit endothelial cell(EC) and hepatocyte apoptosis in vivo and in vitro.

Example 4 Enhanced Islet Graft Survival

The ability of exogenously administered biliverdin or CoPP to enhancesyngeneic islet transplantation was evaluated.

Materials and Methods

Animals. Male DBA/2, B6AF1 and DBA/1 mice 6-8 week of age (Jackson) areused in the experiments. Mice were kept for 2 weeks at 4 mice/cage andfed normal laboratory chow before using for the experiment.

Treatment protocol. Recipients were rendered diabetic usingstreptozotocin (STZ, 225 mg/kg,). Five days after STZ administration,animals with two consecutive blood glucose levels exceeding 350 mg/dlare used as recipients. Islets (500) are transplanted under the kidneycapsule of the recipients.

Reagents. Cobalt protoporphyrin (COPPIX, Porphyrin Products, Logan,Utah), biliverdin dihydrochloride and bilirubin (ICN Biomedicals Inc.,OH) were dissolved in a small amount of 0.2 M NaOH, subsequentlyadjusted to a pH of 7.4 with 1 M HCl, and diluted in 1×PBS. The stocksolutions (COPPIX=2 mg/ml, biliverdin=3.2 mg/ml, bilirubin=0.5 mg/ml)were aliquoted and kept at −70° C. until used. Light exposure waslimited as much as possible.

Induction of HO-1/Administration of biliverdinlbilirubin. When given tothe donor, CoPPIX (20 mg/kg) was given 24 hours before islet isolationto induce HO-1 expression. To verify HO-1 expression in different organsafter CoPP treatment, tissue samples are collected 24 hours aftertreatment and snap frozen in liquid nitrogen. Immunohistologicalanalysis of HO-1 expression is performed using an anti-HO-1 antibody.CoPPIX (20 mg/kg) was administrated to the recipient intraperitoneally(i.p.) on day-1, 1, 3, 5 and 7. Biliverdin (50 μmol/kg) or bilirubin(8.5 μmol/kg) was given intraperitoneally to the donor one hour beforeislet isolation. For the recipient, biliverdin (50 μmol/kg) or bilirubin(8.5 mmol/kg) was administered intraperitoneally either daily or twiceper day from day-1 until day 13. No further treatment was givenafterwards.

Measurement of Glucose level. Glucose levels were tested twice weeklyafter transplantation. Glucose levels of <200 mg/dl were considerednormoglycemic. Grafts were considered rejected when two consecutiveglucose levels were >300 mg/dl.

Tolerance test. The first graft was removed from a number of animalsthat had islets surviving long-term (longer than 100 days) by doing anephrectomy. Islets syngeneic with the original donor were transplantedunder the contralateral kidney capsule without further treatment. Ifthose second transplanted islets also survived longer than 100 dayswithout further treatment, the recipients were considered tolerant.Antigen-specific tolerance was assessed by transplanting islets of athird-party donor (DBA/1) that does not share either class I or class IIantigens with the original donor.

Pathology. Hematoxylin and eosin (H&E) and insulin staining wereperformed on grafts that survived long-term (longer than 100 days).

Semi-Quantitative PCR. RNA was extracted using Rneasy™ Mini Kits (QiagenInc., CA, USA) and reverse transcribed into cDNA with the RNA PCR Kit(TaKaRa, PanVera, Madison, Wis., USA). A total of 2 μg of cDNA wasamplified in a 50 μl reaction mix containing 10 μM dNTPs, 50 μg of5′-prime and 3′-prime oligos, 2.5 U of LA-Taq polymerase (TaKaRa) andMgCl₂, specific to each primer pair used. The primers for murine andhuman HO-1 (372 bp) (5′: TGA AGG AGG CCA CCA AGG AGG T (SEQ ID NO:1);3′: AGG TCA CCC AGG TAG CGG GT (SEQ ID NO:2)) and β-actin (525 bp) (5′:GCC ATC CTG CGT CTG GAC CTG G (SEQ ID NO:3); 3′: TAC TCC TGC TTG CTG ATCCAC A (SEQ ID NO:4)) were obtained from LifeTechnologies, NY, USA. PCRreactions were performed after a 4 min denaturation at 94° C. arepeating the cycle 94° C., 55° C. and 72° C. each for 1 min for numberof cycles specific for each primer pair in a Peltier Termal CyclerPTC-200 (MJ Research, Las Vegas, Nev., USA). PCR products (10-20 μl)were analyzed in an ethidium bromide-stained 1% agarose gel.

Results

HO-1 in allogeneic islet transplantation. When CoPP was given to thedonor animal, HO-1 expression was observed in several different tissuessuch as heart, kidney, pancreas, liver and spleen. HO-1 expression inisolated islets was verified by semi-quantitative PCR analysis. Islettransplantation was done from DBA/2 (H-2^(d)) to B6AF1 (H-2^(b/a)) mice.Subsequently, HO-1 was induced with CoPP for allogeneic transplantation.HO-1 was induced in the donor from one day before taking the islets andin the recipient on days −1, 1, 3, 5 and 7 with regard totransplantation. This protocol led to prolongation of survival ingeneral but also to some islets surviving long-term (longer than 100days) despite the absence of any treatment after day 7 aftertransplantation. In addition, inducing HO-1 only in the donor withouttreatment of the recipient led not only to prolonged survival andfunction of the islets in the recipient but also to an occasionallong-term surviving graft. Survival results with treatment of the donorand recipient and the recipient alone are also shown (FIG. 16). In thetolerance test, all five animals that had been transplanted withsyngeneic islet (DBA/2) accepted the 2^(nd) graft indefinitely (longerthan 100 days) while the third party grafts (n=3) were rejected rapidly.

Induction of long-term survival of allogeneic islets by administrationof biliverdin or bilirubin. Administration of biliverdin or bilirubin tothe DBA/2 donor and B6AF1 recipient led to 66.7% to 100% long-termsurvival, even though there was no treatment given after day 13post-transplantation. The half-life of bilirubin after administration ofbiliverdin was only 2.5 to 3.5 hours. Thus, giving one dose of bilirubinto the donor and recipient was compared to giving two (every twelvehours) doses per day. The single dose led to prolongation of survival ofthe islets and 50% long-term survival while 2 dose led to 100% long-termsurvival. Treatment of the recipient only with biliverdin or bilirubinalso led to a significant percentage of long-term surviving islets (FIG.17). Antigen specific tolerance was also seen in some of the long-termsurvival animals treated with biliverdin or bilirubin.

The ability of exogenously administered biliverdin to protecttransplanted islet was evaluated in the minimal mass model. Syngeneicislet transplantation was carried out in the DBA/2 mouse. Islets (140)were transplanted under the kidney capsule of diabetic mice. Testanimals were treated with 50 μmol/kg biliverdin twice daily from day-1until day 7. The controls were treated with vehicle only. Biliverdintreated animals returned to normoglycemia in 7.7±5.5 days; animals incontrol group remain hyperglycemic (>52 days).

Conclusion

Treatment of the donor and/or recipient of a syngeneic islet transplantwith biliverdin or CoPP enhances graft survival and successful treatmentof diabetic hyperglycemia.

Example 5 Inhibition of Neointimal Formation

The effect of administration of CoPP or biliverdin on formation ofneointimal tissue was evaluated.

Materials and Methods

Animals. Adult male Lewis Rats (LEW/CrIBR; Charles River Laboratories,Wilmington, Mass.) weighing 350-400 g were used in the balloon injurymodel. Animals were housed in accordance with the guidelines from theAmerican Association for Laboratory Animal Care and research protocolswere approved by the institutional animal care and use committee of theBeth Israel Deaconess Medical Center, Boston, Mass.

Balloon Injury model. After the exposure of the left common carotid andthe left external carotid arteries, a 2F Fogarty catheter (EdwardsLifesciences LLC, Irvine, Calif.) was introduced via the externalcarotid artery and was advanced into the left common carotid artery,inflated to 2 atmospheres of pressure and pulled back to the point ofbifurcation, then deflated. This procedure was repeated three times.

Experimental reagents and design. Biliverdin dihydrochloride (ICNBiomedicals Inc., Aurora, Ohio) was dissolved in a small amount of 0.2 MNaOH, subsequently adjusted to a pH of 7.4 with 1 M HCl and diluted inPBS. The stock solution was kept at −70° C. until used. Light exposurewas limited as much as possible.

Local biliverdin delivery. After proximal ligation of the common carotidartery (CCA) and distal ligation of the internal carotid artery (ICA) apolyethylene catheter was introduced into the external branch of thecarotid artery and the CCA was flushed. 50 μl of PBS or Biliverdindiluted in PBS at concentrations of 1 and 0.1 mM was infused andincubated for 1 hour prior to or immediately after the injury, shadedfrom the light. Biliverdin was removed, the CCA flushed two times with0.9% NaCl, and blood flow was restored through the CCA and ICA.

Systemic biliverdin treatment. Biliverdin was injected intraperitoneallyat a dose of 50 μmol/kg. The first dose was injected 3 hours before, thesecond dose immediately after the surgical procedure.

Results

Histomorphometric analysis. Carotid arteries were harvested 14 daysafter balloon injury, either fixed in 10% formalin and imbedded inparaffin or quick-frozen in 2-methylbutane. Serial sections of 5 μm in adistance of 200 μm were stained with hematoxylin-eosin stain. Images of6 sections of each vessel were taken at a resolution of 768×512 pixelswith a Zeiss microscope (Axioskop™, Zeiss, Iowa City, Iowa). Images wereanalyzed by manual segmentation, tracing intima and media in eachsection. Areas and diameters were calculated by digital imaging software(AxioVision™, Carl Zeiss, Jena, Germany) as number of pixelscorresponding to those areas and diameters. Intima media ratio (area,diameter) and luminal cross-sectional area narrowing were used to assessneointimal formation. The person in charge of histomorphometric analysiswas blinded to the treatment. 24 sections from each group werestatistically analyzed with StatView® software version 5.0 using ANOVA.As is shown in FIG. 18, local pre-treatment of rat carotid arteriesinstilling biliverdin into the common carotid artery for one hour aswell as systemic treatment with biliverdin at two time pointssignificantly inhibits neointimal formation (arrows) after ballooninjury.

Conclusion

Treatment with biliverdin or CoPP can significantly reduce arterialinjury following balloon injury, e.g., restenosis.

Example 6 Endotoxic Shock

The anti-inflammatory protective effects of biliverdin were evaluated inan animal model of endotoxic shock.

Materials and Methods

Treatment Protocol. Endotoxin (lipopolysaccharide/LPS; Sigma; E. coliserotype 0128:B7; 3 mg/kg, i.v.) was administered to maleSprague-Dawley, resulting in an acute non-lethal inflammation. In rats,a sublethal dose of LPS results in a moderate lung inflammationcharacterized by neutrophil accumulation and protein accumulation in theairspace, both markers of lung inflammation. The pro-inflammatorycytokine TNF-alpha increases very rapidly in the serum, peaking by 60-90minutes. This is followed by increases in IL-10, a prototypicalanti-inflammatory cytokine that peaks 8-12 hr later.

Admizistration of Biliverdin. Biliverdin (Frontier Scientific: preparedin PBS following solubilization in NaOH) was administered i.p. at 50mmol/kg 17 hours prior to, one hour prior to, and eight hours after LPSadministration.

Bronchoalveolar Lavage. A bronchoalveolar lavage (BAL) was performed 24hours after LPS administration. Serum cytokines were measured usingcommercially available ELISA kits (R&D Systems Inc.) per themanufacturer's instructions. Total protein was determined via a standardBradford assay based on a standard curve. BAL was performed usingstandard methods; briefly, a tracheostomy was performed and 8 ml(approximately 35 ml/kg) of PBS was instilled three times. Total cellcount was determined as well as differential analysis of cell type andmorphology via Diff-QuikS Fixative (American Scientific Products)staining of a sample of the lavagate.

Results

Biliverdin administration reduced levels of LPS-induced TNF-alpha (FIG.19) as well as levels of neutrophil (FIG. 20) and protein (FIG. 21)accumulation in the airspace. Biliverdin administration also resulted inan augmentation of the anti-inflammatory cytokine, IL-10 (FIG. 22).

Conclusions

Biliverdin is a potent anti-inflammatory agent, as evidenced by itsability to reduce the inflammatory effects associated with endotoxinadministration in rats.

Example 7 Hepatitis

The effect of treatment with biliverdin was evaluated in a mouse modelof hepatitis.

Materials and Methods

Male mice (C57BL/6J) were administered biliverdin (50 mmol/kg, i.p.) 16hours and 1 hour prior to i.p. injection of a cocktail including 0.3ug/mouse TNF-α (mouse TNF-α; Gibco) and 250 mg/kg, i.p. D-Galactosamine(Sigma), which induces fulminant hepatitis within 6-10 hours. Controlmice received PBS vehicle. Serum samples were taken via cardiac puncture6-8 hours later and analyzed for alanine aminotransferase (ALT) per anALT assay kit from Sigma Chem. Co. following the manufacturer'sdirections.

Results

Biliverdin reduced serum ALT levels by more than 90% as compared tovehicle treated controls (about 1000 IU/ml in control versus 100 IU/mlin treated animals). As a reference, a normal ALT level is 20-30 IU/ml.

Conclusions

Biliverdin treatment is effective in reducing the liver injury andsymptoms associated with hepatitis, including acute hepatitis.

Example 8 Small Intestine Transplantation

The effect of treatment with biliverdin was assessed in an animal modelof small intestine transplantation.

Materials and Methods.

Animals. Inbred male LEW (RT1) rats weighing 200-300 grams werepurchased from Harlan Sprague Dawley, Inc. (Indianapolis, Ind.), andmaintained in a laminar flow animal facility at the University ofPittsburgh. Animals were fed with a standard diet ad libitum.

Small Intestinal Transplantation. Orthotopic small intestinaltransplantation (SITx) was performed in syngeneic Lewis rats. SITx withcaval drainage was performed using a previously described procedure(Murase et al., in Handbook of Animal Models in TransplantationResearch, Cramer et al., Eds. CRC Press, Boca Raton, Fla., pp. 203-213(1994)). The entire donor small intestine from the ligament of Treitz tothe ileocecal valve was isolated on a vascular pedicle consisting of theportal vein and the superior mesenteric artery in community with asegment of aorta. The graft was perfused via the aortic segment with 5ml chilled Ringer's lactate solution, and the intestinal lumen wasirrigated with 20 ml of cold saline solution containing 0.5%neomycin-sulfate (Sigma, St. Louis, Mo.). End-to-side anastomosesbetween the graft aorta and the recipient infrarenal aorta, and betweenthe graft portal vein and recipient vena cava, were performed with 10-0Novafil™ suture. The cold ischemic time was 1 hour. The entire recipientintestine was removed and the enteric continuity was restored byproximal and distal end-to-end intestinal anastomoses. Recipient animalswere given 20 mg/day prophylactic cefamandole nafate for 3 postoperativedays. Transplanted recipients were given water 3 hours after surgery,and were fed 24 hours after surgery.

SYBR green real time RT-PCR. The effects of administration of biliverdinon transplant-induced pro-inflammatory and anti-inflammatory geneexpression were assessed in muscularis extracts by RT-PCR Biliverdin (50mmol/kg, i.p.) was administered to both the donor and recipient threehours preoperatively. The muscularis extemae was collected from normalintestine and transplanted grafts 4 hours postoperatively and snapfrozen in liquid nitrogen. This time point falls within the range ofmaximum inflammatory mediator expression that occurs between 3 and 6hours following abdominal incision. Total RNA extraction was performedusing the guanidium-thiocyanate phenol-chloroform extraction method asdescribed previously (Eskandari et al., Am. J. Clin. Pathol.75(3):367-370, 1997). RNA pellets were resuspended in RNA-secureresuspension solution (Ambion Inc., Austin, Tex.), followed by removalof potentially contaminating DNA by treatment with DNase I (DNA-FreeKit, Ambion Inc., Austin, Tex.). Equal aliquots (5 μg) of total RNA fromeach sample were quantified by spectrophotometry (wavelength 250 nm) andaliquoted at a concentration of 40 ng/μl. Peak mRNA expression wasquantified in duplicate by SYBR Green two-step, real-time RT-PCR. GAPDHwas used as the endogenous reference. Aliquoted RNA was subjected tofirst-strand complementary DNA (cDNA) synthesis using random hexamers(PE applied Biosystems, Foster City, Calif.) and Super Script II™ (LifeTechnologies, Rockville, Md.). Primer sequences were obtained from theliterature or designed according to published sequences (Table 2). A PCRreaction mixture was prepared using SYBR Green PCR Core Reagents (PEApplied Biosystems). Each sample was estimated in duplicate using theconditions recommended by the manufacturer. The reaction was incubatedat 50° C. for 2 min to activate the uracil N′-glycosylase and then for12 min at 95° C. to activate the Amplitaq Gold™ polymerase followed by40 cycles of 95° C. for 15 sec and 60° C. for 1 min on an ABI PRISM7700™ Sequence Detection System (PE Applied Biosystems, Foster City,Calif.). Real-time PCR data were plotted as the ΔR_(n) fluorescencesignal versus the cycle number. An arbitrary threshold was set to themid-linear portion of the log ΔR_(n) cycle plot. The threshold cycle(C_(T)) was defined as the cycle number at which the ΔR_(n) crosses thisthreshold. Quantification of mRNA expression was normalized to GAPDH andcalculated relative to control using the comparative C_(T) method(Schmittgen et al., J. Biochem. Biophys. Methods 46(1-2):69-8, 2000).

To exclude PCR amplification of contaminating genomic DNA, RT-negativecontrols (samples containing RNA that was not reverse transcribed) wereincluded in each PCR reaction. Melting curve analyses were performed foreach reaction to ensure amplification of specific product. In addition,the primers were subjected to gel electrophoresis to confirm the absenceof non-specific bands and to confirm that the amplicons were of thecorrect size. Efficiency of PCR-amplification of target cDNA wasexamined to measure colinearity of dilution. Serial 3-fold dilutions ofcDNA were performed in triplicate. Standard curves were generated byplotting CT value against relative input copy number. Slopes of thestandard curves of −3.2±0.3 with correlation coefficients of 0.99 wereconsidered to be acceptable, having corresponding efficiencies of100±10%. TABLE 2 Primer summary SEQ ID Primer Sequence 5′ to 3′ NOSource GAPDH ATGGCACAGTCAAGGCTGAGA  9 NM_017008 CGCTCCTGGAAGATGGTGAT 10IL-6 GCCCTTCAGGAACAGCTATGA 11 M26744 TGTCAACAACATCAGTCCCAAGA 12 IL-1βCACCTCTAAGCAGAGCACAG 13 Li & Wang, Brain Research GGGTTCCATGGTGAAGTCAAC14 Protocols 2000; 5, 211-217 TNFα GGTGATCGGTCCCAACAAGGA 15 Fink et al.Nature Med 1998; 4; CACGCTGGCTCAGCCACTC 16 1329-1333. ICAM-1CGTGGCGTCCATTTACACCT 17 NM_012967 TTAGGGCCTCCTCCTGAGC 18 iNOSGGAGAGATTTTTCACGACACCC 19 NM_012611 CCATGCATAATTTGGACTTGCA 20 COX-2CTCTGCGATGCTCTTCCGAG 21 AF233596 AAGGATTTGCTGCATGGCTG 22 IL-10TGCAACAGCTCAGCGCA 23 Harness et al., J. Neurol. Sci.GTCACAGCTTTCGAGAGACTGGAA 24 2001; 187, 7-16.

Motility Studies. The effect of administration of biliverdin ontreatment on intestinal dysmotility in transplanted grafts was assessedboth in vitro and in vivo. Tissues were harvested 24 or 48 hourspost-operatively, which have been shown to be time points during whichtransplant-induced dysmotility peaks. In vitro circular musclemechanical activity was measured as previously described (Eskandari etal., Am. J. Physiol. 273(3 Pt 1):G727-34, 1997). Rats were anesthetizedand killed by exsanguination 24 hours post-operatively. A segment ofmid-jejunum was pinned in a Sylgaard™ lined dissecting dish containingpre-oxygenated Krebs-Ringer-bicarbonate buffer (KRB; in mM: 137.4 Na⁺,5.9 K⁺, 2.5 Ca²⁺, 1.2 Mg²⁺, 134 Cl⁻, 15.5 HCO₃ ⁻, 1.2 H₂PO₄ ⁻, and 11.5glucose) that was equilibrated with 97% O₂/3% CO₂. The intestine wasopened along the mesenteric border and the mucosa removed by strippingwith fine forceps. Full-thickness strips of muscularis (1×6 mm) were cutparallel to the circular muscle layer. Muscle strips were mounted instandard horizontal mechanical organ chambers that were continuouslysuperfused with pre-oxygenated KRB maintained at 37° C. One end of eachstrip was attached by ligature to a fixed post and the other to anisometric force transducer (WPI, Sarasota, Fla.). Strips were allowed toequilibrate for 1 hour, after which they were incrementally stretched tothe length at which maximal spontaneous contraction occurred (L_(o)).After a second equilibration period of 30 minutes,contractility-response curves were generated by exposing the tissues toincreasing concentrations of the muscarinic agonist bethanechol (0.3 to300 μM) for 10 minutes, followed by a 10-minute wash period. Contractileactivity was calculated by integrating the area under the trace,normalized by converting the weight and length of the strip to squaremillimeters of tissue (1.03 mg/mm²), and reported as g/s/mm².

Measurement of Intestinal Blood Flow. Intestinal microvascular bloodflow was monitored by placing the flat probe of a laser Dopplerflowmeter (BLF 21D, Transonic Systems, Ithaca, N.Y.) on the serosalsurface of the graft jejunum and ileum adjacent to the mesentericborder. Blood flows in SMA and marginal artery (MA) were also analyzed.

Measurement of Intestinal Blood Flow. Microvascular blood flow for ingrafts preserved for 6 hours was measured using a laser Dopplerflowmeter (BLF 21D, Transonic Systems, Ithaca, N.Y.) equipped with aflat probe. Intestinal microvascular blood flow was monitored by placingthe probe on the serosal surface of the graft jejunum and ileum adjacentto the mesenteric border. Blood flow in the superior mesenteric artery(SMA) and marginal artery (MA) were also measured.

Graft permeability. The preparation of everted gut sacs was performed inice-cold modified Krebs-Henseleit bicarbonate buffer [KHBB (pH 7.4)]consisting of 10 mM HEPES, 137.0 mM NaCl, 5.36 mM KCl, 4.17 mM NaHCO3,0.34 mM Na2HPO4, 0.44 mM KH2PO4, 0.41 mM MgSO47H₂O, 0.49 mM MgC126H₂₀,1.26 mM CaCl₂, and 19.45 mM glucose. One end of the gut segment wasligated with 4-0 silk. The resulting gut sac was everted using a thinplastic rod. A groove was cut into the tip of a 5 ml plastic syringe and1.5 mL of KHBB was drawn into the syringe. The open end of the evertedgut sac was secured to the groove using 4-0 silk, and the intestine wasgently distended by injecting the KHBB from the syringe. The sac wassuspended in a beaker holding 80 mL of a solution of KHBB maintained at37° C. and containing fluorescein-isothiocyanate dextran (average M.W.4000 Da; FD4; 20 mg/mL). The bathing solution was aerated by gentlybubbling with a gas mixture containing 95% O₂ and 5% CO₂. At thebeginning of the experiment, 1.0 mL of the bathing solution was removedto measure the initial concentration of FD4. Following a 30 minincubation, the length of the sac was measured, and 1.0 mL of the fluidwithin the gut sac was collected. The samples were cleared bycentifugation at 1,000 rpm for 10 min at 4° C. Subsequently, 300 ul ofthe supernatant was diluted with 3.0 mL of phosphate buffered saline,and the fluorescence of the solution was measured using a Perkin-ElmerLS-50 fluorescence spectrophotometer (Palo Alto, Calif.) at anexcitation wave length of 492 nm (slit width=10.0 nm) and an emissionwavelength of 515 nm (slit width=10.0 mm).

Detection of Serum Mediators. Serum samples from the recipientstransplanted with preserved intestine were taken at 3 and 12 hours afterreperfusion and stored at −800 until evaluation. Serum IL-6 wasdetermined using a rat enzyme-linked immunoassay (ELISA) kits asdescribed by the manufacturer (R & D Systems, Inc., Cambridge, Mass.).To monitor the production of nitric oxide, the stable end products of NOmetabolism, serum nitrite/nitrate levels, were measured 12 hours afterengraftment using a commercially available test kit (Cayman, Ann Arbor,Mich.). In this assay system, nitrate is reduced to nitrite usingnitrate reductase, and the nitrite concentration of the sample issubsequently measured using the Griess reaction.

Data Analysis. Results are expressed as mean plus or minus the standarderror of the mean (SEM). Statistical analysis was performed usingStudent's t test or analysis of various (ANOVA) where appropriate. Aprobability level of p<0.05 was considered statistically significant.

Results

SITx without Preservation (Minor Injury)

Serum bilirubin levels and BVR expression in the graft. To assess howfast injected biliverdin (BV) is metabolized into bilirubin, sequentialserum bilirubin levels were analyzed. Before BV treatment, serumbilirubin levels were undetectable. Thirty minutes after BV injectioni.p., serum bilirubin levels reached a peak of 1.07±0.5 mg/dl comparedto those of normal animals. By 2 hours after injection i.p., bilirubinlevels returned to normal.

Circular muscle contractility. To determine the direct effects of SITxand BV on the muscular apparatus, the effects of SITx with and withoutBV treatment were investigated on spontaneous and bethanechol-stimulatedjejunal circular muscle contractility using in vitro organ bathexperiments. Tissues were harvested 24 hours after transplantation ofthe intestinal graft, a time point when intestinal motility associatedwith SITx is known to be maximally suppressed (Schwarz et al., Surgery131:413-423 (2002)).

Control animals treated with BV demonstrated no change in theirspontaneous muscle contractile activity. SITx results in a significantdecrease in spontaneous muscle contractile activity, however, jejunalmuscle strips harvested from grafts transplanted into recipient animalstreated with BV demonstrated significantly greater spontaneouscontractile activity as compared to untreated transplants.

The addition of bethanechol (0.3 to 300 μM) to the bathing superfusateelicited a concentration-dependent increase in circular musclecontractility. Control muscles from untreated and BV treated animalsexhibited similar robust phasic and tonic contractions to bethanechol(100 μM), while muscles from the untreated transplanted intestinegenerated approximately 51% less contractility in response tobethanechol (1.7=0.4 g/mm2/s). However, bethanechol-stimulated musclecontractility generated by BV treated animals was significantly improvedover the untreated graft muscles. These observations were reflectedthroughout the generation of the complete integrated contractilebethanechol dose-response curves for each of the four groups of animals.As shown in FIG. 25, BV therapy reduced the transplant-inducedsuppression in muscle contractility, restoring the muscle's response topre-transplant levels.

Leukocyte Recruitment. Cellular inflammatory events in the smallintestinal muscularis were characterized 24 hours after SITx.Myeloperoxidase (MPO) activity, as determined by Hanker-Yateshistochemistry, was used to quantify the polymorphonuclear neutrophil(PMN) infiltrate in tissues from control and transplanted animals, withand without BV treatment. In unoperated controls with saline,MPO-positive cells were rare. BV injection into normal animals decreasedMPO positive cells extravasation to 3.9±1.3 cells per ×200 field, butdid not reach significance compared to saline controls. SITx resulted ina significant recruitment of PMNs into the intestinal muscularis. BVtreatment significantly decreased the mean number of MPO positive cells.

Molecular Inflammatory Responses. Four hours following reperfusion, mRNAlevels of various prototypical inflammatory mediators were determined byquantitative analysis.

Real time RT-PCR analysis revealed a significant increase in mRNAexpression for the inflammation-related cytokines, IL-6, IL-10, TNFα andIL-1β in graft muscularis externa extracts 4 hours after reperfusion,when compared to unoperated saline-controls (FIG. 23A-D).

In graft muscularis extracts of recipient rats treated with BV, themean-comparative expression of IL-6 and IL-1b expression was reduced onaverage by 24% (p=0.0099, N=5) and 30% (p=0.0040, N=5), respectively,compared to the saline-treated transplanted and reperfused graft at 4hours (FIGS. 23A and 23D). However, unlike IL-6 and IL-1b, BV treatmentdid not significantly change the upregulation of TNF-α or IL-10 causedby transplantation (FIGS. 23B and 23C. BV treatment of unoperatedanimals also did not alter the basal mRNA expression of any of thecytokines.

Gene expression of inducible nitric oxide synthase (iNOS) andcyclooxygenase (COX-2) were quantified by real time RT-PCR. The resultsshowed that both INOS and COX-2, enzymes of the puissant smooth muscleinhibitors nitric oxide and prostanoids, were significantly upregulatedin the muscularis of the transplanted grafts 70.4-fold and 5.2-fold,respectively (FIG. 24A-B). The mean relative mRNA expression of bothenzymes was reduced by approximately 50% in BV treated rats (p=0.015 andp=0.032, N=5 each). BV treatment of unoperated control animals did notalter the mRNA expression of iNOS or COX-2. ICAM-1 gene expression, anadhesion molecule that plays an important role in the recruitment ofcirculating inflammatory cells into inflamed tissues, was alsosignificantly increased 6.1±3.8-fold compared with controls. BVtreatment significantly reduced ICAM-1 expression in the graft up to 30%(p=0.020) (FIG. 23C). BV treatment significantly reduced MnSODexpression in the graft as well (FIG. 23D).

HO-1 Induction During Intestinal I/R Injury. Since HO-1 regulates hemecatalysis and BV production, exogenously provided BV may influenceendogenous HO-1 induction in BV-treated recipients. In the normalintestine, HO-1 protein expression was absent. Ischemia/reperfusioninjury was associated with a gradual increase of HO-1 expression inair-treated grafts, reaching maximum level between 6 and 24 hoursfollowing reperfusion. BV treatment did not have a significant effect onthe intestinal HO-1 production.

SITx with 6 Hours Preservation (Severe Injury)

Antioxidant capacity after BV treatment. the total antioxidant capacityor antioxidant power within the graft specimens was quantitativelymeasured. Antioxidant levels in the graft were detected using TotalAntioxidant Power® (Oxford Biomedical Research, Oxford, Mich.),according to the manufacturer's instructions. In this procedure, theevaluation of the antioxidant level in a sample is detected byevaluation of Cu⁺ derived from Cu++by the combined action of allantioxidants present in the sample. As is shown in FIG. 27, BV treatmentincreased the antioxidant capacity of the transplanted intestine tonormal levels.

Serum inflammatory mediators. The decreased expression of IL-6 and iNOSmRNA (FIGS. 23A and 24B) was also reflected in protein production aftertransplantation of cold-preserved grafts; saline and BV treated controlsserum IL-6 concentrations were low. Transplantation caused a significantincrease in serum IL-6 protein concentrations (5131.4±3169.1 pg/mL) 3hours after engraftment and the serum IL-6 increase was significantlyless in animals that had received BV therapy (1652±306.9 pg/mL,p=0.0347). Because of the importance of nitric oxide as a regulator ofgastrointestinal motility, the molecular expression of iNOS was followedby measuring nitric oxide metabolites in the serum of the transplantedanimals. In saline and BV treated controls, mean serum nitrite/nitrate(NO) levels were 17.3±7.4 and 18.4±3.2 μM, respectively. SITx resultedin a significant elevation of serum NO products to 34.4±15.2 μM 12 hoursafter engraftment. BV treatment significantly decreasedtransplantation-induced serum NO levels by 53% to 18.2±3.9 μM.

Graft permeability. Loss of intestinal barrier function causes increaseof graft permeability. Graft permeability was determined by the evertedgut sac method using 4 Kd Fluorescein isothiocyanate dextran. A strildngincrease in permeability was seen in untreated grafts (1.44±1.0ml/cm/min). In BV treated grafts, there was a minimal increase ofintestinal permeability, or about a 60% inhibition of the increase, to2.82% 1.3 ml/cm/min (FIG. 26A).

Blood microcirculation. Blood flow following transplantation isdramatically decreased. Administration of BV had no effect onmicrovascular blood flow following transplantation (FIG. 26B).

Animal survival. Six hours of cold preservation in UW of the intestinalgraft induced intestinal dysfunction in untreated recipients; 3 out of14 control animals died within 24 hours and an additional 3 animals died5 and 7 days after SITx due to bowel obstruction secondarily tointestinal I/R injury. In contrast, all BV-treated animals recoveredsmoothly from SITx. Overall animal survival for 14 days follow-up was57.1% (8/14) in saline control and 100% (8/8) in BV-treated group(p<0.05).

Conclusions

The above data indicated that BV treatment results in the blunting ofthe proinflammatory responses within the graft intestinal muscularisfollowing transplantation, enhancing small intestine graft function andrecipient survival.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of reducing inflammation in a patient, comprising:identifying a patient suffering from or at risk for inflammation; andadministering to the patient at least one treatment selected from thegroup consisting of: inducing ferritin in the patient; expressingferritin in the patient; and administering a pharmaceutical compositioncomprising HO-1, bilirubin, biliverdin, ferritin, iron, desferoxamine,salicylaldehyde isonicotinoyl hydrazone, iron dextran, or apoferritin tothe patient, in an amount sufficient to reduce inflammation.
 2. Themethod of claim 1, wherein the treatment is inducing ferritin in thepatient.
 3. The method of claim 1, wherein the treatment is expressingferritin in the patient.
 4. The method of claim 1, wherein the treatmentis administering a pharmaceutical composition comprising HO-1 to thepatient.
 5. The method of claim 1, wherein the treatment isadministering a pharmaceutical composition comprising biliverdin to thepatient.
 6. The method of claim 5, wherein the pharmaceuticalcomposition is administered to the patient at a dosage of about 1 to1000 micromoles/kg/day.
 7. The method of claim 6, wherein theinflammation is associated with ulcerative colitis.
 8. The method ofclaim 1, wherein the treatment is administering a pharmaceuticalcomposition comprising bilirubin to the patient.
 9. The method of claim1, wherein the treatment is administering a pharmaceutical compositioncomprising ferritin to the patient.
 10. The method of claim 1, whereinthe treatment is administering a pharmaceutical composition comprisingdesferoxamine (DFO) or salicylaldehyde isonicotinoyl hydrazone (SIH) tothe patient.
 11. The method of claim 1, wherein the treatment isadministering a pharmaceutical composition comprising iron dextran tothe patient.
 12. The method of claim 1, wherein the treatment isadministering a pharmaceutical composition comprising apoferritin to thepatient.
 13. The method of claim 2, wherein the ferritin is induced byadministering iron to the patient.
 14. The method of claim 1, whereinthe inflammation is associated with a condition selected from the groupconsisting of: asthma, adult respiratory distress syndrome, interstitialpulmonary fibrosis, pulmonary emboli, chronic obstructive pulmonarydisease, primary pulmonary hypertension, chronic pulmonary emphysema,congestive heart failure, peripheral vascular disease, stroke,atherosclerosis, ischemia-reperfusion injury, heart attacks,glomerulonephritis, conditions involving inflammation of the kidney,infection of the genitourinary tract, viral and toxic hepatitis,cirrhosis, ileus, necrotizing enterocolitis, specific and non-specificinflammatory bowel disease, rheumatoid arthritis, deficient woundhealing, Alzheimer's disease, Parkinson's disease, graft versus hostdisease, and hemorrhagic, septic, or anaphylactic shock.
 15. The methodof claim 1, wherein the inflammation is inflammation of the heart, lung,liver, spleen, brain, skin, and/or kidney.
 16. The method of claim 1,wherein the inflammation is an inflammatory condition localized in thegastrointestinal tract.
 17. The method of claim 16, wherein theinflammatory condition is selected from the group consisting of: amoebicdysentery, bacillary dysentery, schistosomiasis, campylobacterenterocolitis, yersinia enterocolitis, enterobius vermicularis,radiation enterocolitis, ischaemic colitis, eosinophilicgastroenteritis, ulcerative colitis, indeterminate colitis, and Crohn'sdisease.
 18. The method of claim 17, wherein the inflammatory conditionis ulcerative colitis.
 19. The method of claim 1, further comprising thestep of administering to the patient at least one treatment selectedfrom the group consisting of: inducing HO-1 in the patient; expressingHO-1 in the patient; and administering a pharmaceutical compositioncomprising carbon monoxide to the patient.
 20. The method of claim 1,further comprising the steps of inducing HO-1 in the patient, andadministering a pharmaceutical composition comprising carbon monoxide tothe patient.
 21. A method of transplanting an organ, the methodcomprising: (a) administering to a donor at least one treatment selectedfrom the group consisting of: inducing ferritin in the donor; expressingferritin in the donor; and administering a pharmaceutical compositioncomprising HO-1, bilirubin, biliverdin, ferritin, desferoxamine, irondextran, or apoferritin to the donor; (b) obtaining an organ from thedonor; and (c) transplanting the organ into a recipient, wherein thetreatment administered in step (a) is sufficient to enhance survival orfunction of the organ after transplantation into the recipient.
 22. Amethod of transplanting an organ, the method comprising: (a) providingan organ of a donor; (b) administering to the organ ex vivo at least onetreatment selected from the group consisting of: inducing ferritin inthe organ; expressing ferritin in the organ; and administering apharmaceutical composition comprising HO-1, bilirubin, biliverdin,ferritin, desferoxamine, iron dextran, or apoferritin; and (c)transplanting the organ into a recipient, wherein treatment administeredto the organ in step (b) is sufficient to enhance survival or functionof the organ after transplantation of the organ to the recipient. 23.The method of transplanting an organ, the method comprising: (a)providing an organ from a donor; (b) transplanting the organ into arecipient; and (c) before, during, or after step (b), administering tothe recipient at least one treatment selected from the group consistingof: inducing ferritin in the recipient; expressing ferritin in therecipient; and administering a pharmaceutical composition comprisingHO-1, bilirubin, biliverdin, ferritin, desferoxamine, iron dextran, orapoferritin to the recipient, wherein the treatment administered to therecipient in step (c) is sufficient to enhance survival or function ofthe organ after transplantation of the organ to the recipient.
 24. Themethod of claim 21, further comprising the step of administering to thedonor at least one treatment selected from the group consisting of:inducing HO-1 in the donor; expressing HO-1 in the donor; andadministering a pharmaceutical composition comprising carbon monoxide tothe donor.
 25. The method of claim 21, further comprising the steps ofinducing HO-1 in the donor, and administering a pharmaceuticalcomposition comprising carbon monoxide to the donor.
 26. The method ofclaim 22, further comprising the step of administering to the patient atleast one treatment selected from the group consisting of: inducing HO-1in the organ; expressing HO-1 in the organ; and administering apharmaceutical composition comprising carbon monoxide to the organ. 27.The method of claim 22, further comprising the steps of inducing HO-1 inthe organ and administering a pharmaceutical composition comprisingcarbon monoxide to the organ.
 28. The method of claim 23, furthercomprising the step of administering to the patient at least onetreatment selected from the group consisting of: inducing HO-1 in therecipient; expressing HO—I in the recipient; and administering apharmaceutical composition comprising carbon monoxide to the recipient.29. The method of claim 23, further comprising the steps of inducingHO-1 in the recipient, and administering a pharmaceutical compositioncomprising carbon monoxide to the recipient.
 30. A method of performingangioplasty on a patient, comprising: (a) performing angioplasty on thepatient; and (b) before, during, or after the performing step,administering at least one treatment selected from the group consistingof: inducing ferritin in the patient; expressing ferritin in thepatient; and administering a pharmaceutical composition comprising HO-1,bilirubin, biliverdin, ferritin, desferoxamine, iron dextran, orapoferritin to the patient.
 31. The method of claim 30, furthercomprising the step of administering to the patient at least onetreatment selected from the group consisting of: inducing HO-1 in thepatient; expressing HO-1 in the patient; and administering apharmaceutical composition comprising carbon monoxide to the patient.32. The method of claim 30, further comprising the steps of inducingHO-1 in the patient and administering a pharmaceutical compositioncomprising carbon monoxide to the patient.
 33. A method of performingvascular surgery on a patient, comprising: (a) performing vascularsurgery on the patient; and (b) before, during, or after the performingstep, administering at least one treatment selected from the groupconsisting of: inducing HO-1 or ferritin in the patient; expressing HO—Ior ferritin in the patient; and administering a pharmaceuticalcomposition comprising HO-1, bilirubin, biliverdin, ferritin,desferoxamine, iron dextran, or apoferritin to the patient.
 34. Themethod of claim 33, further comprising the step of administering to thepatient at least one treatment selected from the group consisting of:inducing HO-1 in the patient; expressing HO-1 in the patient; andadministering a pharmaceutical composition comprising carbon monoxide tothe patient.
 35. The method of claim 33, further comprising the steps ofinducing HO-1 in the patient and administering a pharmaceuticalcomposition comprising carbon monoxide in the patient.
 36. A method oftreating a cellular proliferative and/or differentiative disorder in apatient, comprising: identifying a patient suffering from or at risk fora cellular proliferative and/or differentiative disorder; andadministering to the patient at least one treatment selected from thegroup consisting of: inducing ferritin in the patient; expressingferritin in the patient; and administering a pharmaceutical compositioncomprising HO-1, bilirubin, biliverdin, ferritin, iron, desferoxamine,iron dextran, or apoferritin to the patient, in an amount sufficient totreat the cellular proliferative and/or differentiative disorder. 37.The method of claim 36, further comprising the step of administering tothe patient at least one treatment selected from the group consistingof: inducing HO-1 in the patient; expressing HO-1 in the patient; andadministering a pharmaceutical composition comprising carbon monoxide tothe patient.
 38. The method of claim 36, further comprising the steps ofinducing HO-1 in the patient and administering a pharmaceuticalcomposition comprising carbon monoxide in the patient.
 39. A method ofreducing the effects of ischemia in a patient, comprising: identifying apatient suffering from or at risk for ischemia; and administering to thepatient at least one treatment selected from the group consisting of:inducing ferritin in the patient; expressing ferritin in the patient;and administering a pharmaceutical composition comprising HO-1,bilirubin, biliverdin, ferritin, iron, desferoxamine, iron dextran, orapoferritin to the patient, in an amount sufficient to reduce theeffects of ischemia.
 40. The method of claim 40, further comprising thestep of administering to the patient at least one treatment selectedfrom the group consisting of: inducing HO-1 in the patient; expressingHO-1 in the patient; and administering a pharmaceutical compositioncomprising carbon monoxide to the patient.
 41. The method of claim 40,further comprising the steps of inducing HO-1 in the patient, andadministering a pharmaceutical composition comprising carbon monoxide tothe patient.
 42. A method of treating atherosclerosis, the methodcomprising: identifying an individual suffering from or at riskatherosclerosis; and administering to the individual a pharmaceuticalcomposition comprising biliverdin, bilirubin or a mixture thereof in anamount sufficient to treat atherosclerosis.
 43. The method of claim 42,wherein the pharmaceutical composition is suitable for oraladministration.
 44. The method of claim 43, wherein the pharmaceuticalcomposition is in tablet or capsule form.
 45. The method of claim 42,wherein the pharmaceutical composition is a controlled releaseformulation.
 46. The method of claim 42, wherein the pharmaceuticalcomposition is administered to the individual at least once per day. 47.The method of claim 42, wherein the pharmaceutical composition isadministered to the individual several times per day.
 48. The method ofclaim 42, wherein the pharmaceutical composition comprises biliverdin.49. The method of claim 48, wherein the pharmaceutical composition isadministered to the individual in a dose of about 1 to 1000 μmol/kg/day.50. The method of claim 42, wherein the pharmaceutical compositioncomprises bilirubin.
 51. The method of claim 50, wherein thepharmaceutical composition is administered to the individual in a doseof about 1 to 1000 mg/kg/day.
 52. The method of claim 42, wherein thepharmaceutical composition comprises a mixture of bilirubin andbiliverdin.
 53. The method of claim 42, wherein the pharmaceuticalcomposition is suitable for transdermal or transmucosal administration.54. A pharmaceutical composition in tablet or capsule form comprisingbiliverdin, bilirubin or a mixture thereof.
 55. The pharmaceuticalcomposition of claim 54, wherein the pharmaceutical composition is acontrolled release formulation.
 56. A pharmaceutical composition in aform suitable for transmucosal or transdermal administration comprisingbiliverdin, bilirubin or a mixture thereof.
 57. The pharmaceuticalcomposition of claim 56, wherein the composition is in nasal spray orsuppository form.
 58. The pharmaceutical composition of claim 56,wherein the composition is in ointment, salve, gel or cream form.